Oral delivery of oligonucleotides

Abstract

To provide new and improved methods for oral delivery of siRNA molecules in vivo, to achieve and enhance the therapeutic potential of iRNA agents.SOLUTION: One aspect of the present invention relates to an oral formulation for reducing or inhibiting the expression of a target gene in a subject, comprising: a) double stranded iRNA agent comprising an antisense strand which is complementary to a target gene, a sense strand which is complementary to the antisense strand, 2'-OMe modifications to more than 15, more than 20, more than 25, or more than 30 nucleotides, and a carbohydrate-based ligand conjugated to at least one strand optionally via a linker or carrier; and b) a penetration enhancer. Another aspect of the invention relates to a method of gene silencing, comprising orally administering the oral formulation to a subject in need thereof.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 849,605, filed May 17, 2019; and U.S. Provisional Patent Application No. 62 / 911,512, filed October 7, 2019, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to the field of oral delivery using ligand-conjugated oligonucleotides. [Background technology]

[0003] To date, efficient and effective oral delivery of iRNA agents has remained a challenge. Despite ongoing efforts in this field, sufficient oral bioavailability and resulting therapeutic efficacy of intact functional oligonucleotides (the fraction of the administered dose that reaches the systemic circulation) have yet to be demonstrated, limiting the use of siRNA-based therapies.

[0004] Oral administration of hydrophilic macromolecules with molecular weights (MW) greater than 1000 Da, such as iRNA agents, is particularly problematic due to multiple challenges, including degradation at low gastric pH or by gastrointestinal enzymes, low permeability across gastrointestinal membranes, and first-pass metabolism (the process of absorption into the liver and intestinal wall).

[0005] One possible solution to the permeability problem could be the use of intestinal permeation enhancers for oral drug delivery. For example, it has been reported that medium-chain fatty acids such as sodium caprate (C10) improve cellular permeability in the delivery of FITC-dextran (MW 4K), polysucrose (MW 15K), and insulin (see Non-Patent Document 1). However, in that report, the effect was significant only for substances with a molecular weight less than 1.2K; permeation enhancement would not have resulted in a significant increase in the absorbed dose fraction for larger molecules. Sulcaprozate sodium (SNAC) and sodium caprate (C10) have been used as permeation enhancers for oral delivery of peptides and proteins (see Non-Patent Document 2). However, to date, formulations and appropriate dosing regimens using permeation enhancers for oral delivery of macromolecules have not yet been clarified. Efficient and effective oral delivery of iRNA agents in clinically relevant dosing regimens has not yet been established. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Cano-Cebrian et al.,Current Drug Delivery 2(1):9-22(2005) [Non-patent document 2] Twarog et al.,Phamaceutics 11(2):E78(2019) Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a continuing need for new and improved methods for oral delivery of siRNA molecules in vivo to achieve and enhance the therapeutic potential of iRNA agents. [Means for solving the problem]

[0008] One aspect of the invention relates to an oral formulation for reducing or inhibiting expression of a target gene in a subject. The oral formulation includes a) a double-stranded iRNA agent and b) a penetration enhancer. The double-stranded iRNA agent includes an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and a carbohydrate-based ligand conjugated to at least one of the strands, optionally via a linker or carrier. The double-stranded iRNA agent also includes 2'-OMe modifications to more than 15, more than 20, more than 25, or more than 30 nucleotides.

[0009] In some embodiments, the concentration of the penetration enhancer in the oral formulation is about 200 mM or less, e.g., about 150 mM or less, about 100 mM or less, about 80 mM or less, about 60 mM or less, about 50 mM or less, about 45 mM or less, about 40 mM or less, about 35 mM or less, or about 30 mM or less.

[0010] A carbohydrate-based ligand can be conjugated to an iRNA agent via a direct bond to the iRNA agent's ribosugar, or alternatively, a carbohydrate-based ligand can be conjugated to an iRNA agent via a linker or carrier.

[0011] In certain embodiments, the carbohydrate-based ligand is conjugated to the iRNA agent via one or more linkers (tethers).

[0012] In some embodiments, the carbohydrate-based ligand is conjugated to the double-stranded iRNA agent via a linker that includes an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0013] In some embodiments, at least one of the linkers (tethers) is a redox-cleavable linker (a reductively cleavable linker; e.g., a disulfide group), an acidic cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase-cleavable linker (e.g., an ester group), a phosphatase-cleavable linker (e.g., a phosphate group), or a peptidase-cleavable linker (e.g., a peptide bond).

[0014] In other embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0015] In certain embodiments, the carbohydrate-based ligand is conjugated to the double-stranded iRNA agent via a carrier that replaces one or more nucleotides. The carrier can be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol or diethanolamine backbone.

[0016] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the double-stranded iRNA agent.

[0017] In other embodiments, the carrier replaces a nucleotide at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby functioning as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine. For example, the carrier can be cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0018] In some embodiments, the carbohydrate-based ligand is attached to the end of the sense or antisense strand. In one embodiment, the carbohydrate-based ligand is attached to the 3'-end of the antisense strand. In one embodiment, the carbohydrate-based ligand is attached to the 5'-end of the antisense strand. In one embodiment, the carbohydrate-based ligand is attached to the 5'-end of the sense strand. In one embodiment, the carbohydrate-based ligand is attached to the 3'-end of the sense strand.

[0019] In some embodiments, the carbohydrate-based ligand is D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent fucose, a glycosylated polyamino acid, or a lectin.

[0020] In some embodiments, the carbohydrate-based ligand is an ASGPR ligand. For example, the ASGPR ligand may be linked to a bivalent or trivalent branched linker, such as: [ka] In one embodiment, the ASGPR ligand is attached to the 3' or 5' end of the sense strand.

[0021] In some embodiments, the sense and antisense strands of a double-stranded iRNA agent are each 15-30 nucleotides in length. In one embodiment, the sense and antisense strands of a double-stranded iRNA agent are each 19-25 nucleotides in length. In one embodiment, the sense and antisense strands of a double-stranded iRNA agent are each 21-23 nucleotides in length.

[0022] In some embodiments, the double-stranded iRNA agent includes a single-stranded overhang on at least one of its termini, e.g., a 3' and / or 5' overhang 1-10 nucleotides in length, e.g., an overhang of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, both strands have at least one stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length on at least one of its termini.

[0023] In some embodiments, a double-stranded iRNA agent may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. In one embodiment, a double-stranded iRNA agent includes a 3' overhang at the 3' end of the antisense strand, and optionally a blunt end at the 5' end of the antisense strand. In one embodiment, a double-stranded iRNA agent has a 5' overhang at the 5' end of the sense strand, and optionally a blunt end at the 5' end of the antisense strand. In one embodiment, a double-stranded iRNA agent has two blunt ends at both ends of the iRNA duplex.

[0024] In one embodiment, the sense strand of the double-stranded iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide-long single-stranded overhang at the 3' end.

[0025] In some embodiments, a carbohydrate-based ligand is conjugated to a nucleobase, sugar moiety, or internucleoside linkage of a double-stranded iRNA agent.

[0026] In some embodiments, the double-stranded iRNA agent further comprises a phosphate mimic at the 5' end of either the sense strand or the antisense strand, or both strands. In one embodiment, the phosphate mimic is at the 5' end of the antisense strand.

[0027] The phosphate mimics may be 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl [ka] In one embodiment, the phosphate mimic is 5'-vinylphosphonate (VP). 5'-VP is the 5'-E-VP isomer (i.e., trans-vinyl phosphate, [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ) or a mixture thereof.

[0028] In some embodiments, the 5' end of either the sense strand or the antisense strand or both strands of a double-stranded iRNA agent does not contain a 5'-vinylphosphonate (VP).

[0029] In some embodiments, the double-stranded iRNA agent further comprises at least one terminal chiral phosphorus atom.

[0030] Site-specific chiral modifications of internucleotide linkages can occur at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the strand. This is referred to herein as a "terminal" chiral modification. Terminal modifications can occur at the 3'- or 5'-end position in the terminal region, for example, at the terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the strand. Chiral modifications can occur in the sense strand, the antisense strand, or both the sense and antisense strands. Each chirally pure phosphorus atom can be in either the Rp or Sp configuration, and combinations thereof. Further details regarding chiral modifications and chirally modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, entitled "Chirally-Modified Double-Stranded RNA Agents," the entire contents of which are incorporated herein by reference.

[0031] In some embodiments, the double-stranded iRNA agent further includes a terminal chiral modification present at the first internucleotide linkage at the 3'-end of the antisense strand, having the bound phosphorus atom in the Sp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, having the bound phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, having the bound phosphorus atom in either the Rp or Sp configuration.

[0032] In one embodiment, the double-stranded iRNA agent further includes a terminal chiral modification present at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linked phosphorus atom in the Sp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, with the linked phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, with the linked phosphorus atom in either the Rp or Sp configuration.

[0033] In one embodiment, the double-stranded iRNA agent further includes a terminal chiral modification present at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand, with the linked phosphorus atom in the Sp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, with the linked phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, with the linked phosphorus atom in either the Rp or Sp configuration.

[0034] In one embodiment, the double-stranded iRNA agent further includes a terminal chiral modification present at the first and second internucleotide linkages at the 3'-end of the antisense strand, with the linking phosphorus atom in the Sp configuration; a terminal chiral modification present at the third internucleotide linkage at the 3'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification present at the first internucleotide linkage at the 5'-end of the sense strand, with the linking phosphorus atom in either the Rp or Sp configuration.

[0035] In one embodiment, the double-stranded iRNA agent further includes terminal chiral modifications present at the first and second internucleotide linkages at the 3'-end of the antisense strand, having the linked phosphorus atom in the Sp configuration; terminal chiral modifications present at the first and second internucleotide linkages at the 5'-end of the antisense strand, having the linked phosphorus atom in the Rp configuration; and terminal chiral modifications present at the first internucleotide linkage at the 5'-end of the sense strand, having the linked phosphorus atom in either the Rp or Sp configuration.

[0036] In some embodiments, the double-stranded iRNA agent has at least two blocks of two consecutive phosphorothioate or methylphosphonate internucleotide linkage modifications.

[0037] In some embodiments, the antisense strand comprises two blocks of 1, 2, or 3 phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0038] In some embodiments, the antisense strand comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, counting from the 5'-end of the antisense strand. In some embodiments, the sense strand comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand, counting from the 5'-end of the sense strand. In one embodiment, the antisense strand comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, counting from the 5'-end of the antisense strand; and the sense strand comprises at least two consecutive phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand, counting from the 5'-end of the sense strand.

[0039] In some embodiments, a double-stranded iRNA agent has at least 15 nucleotides with a 2'-modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-O-allyl, ENA, and BNA / LNA modifications. In one embodiment, a double-stranded iRNA agent has at least 15 nucleotides with a 2'-O-alkyl modification, such as a 2'-O-methyl modification. A double-stranded iRNA agent can have 2'-OMe modifications on more than 15, more than 20, more than 25, or more than 30 nucleotides.

[0040] In some embodiments, the double-stranded iRNA agent includes at least two blocks of two consecutive phosphorothioate or methylphosphonate internucleotide linkage modifications.

[0041] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of all nucleotides of a double-stranded iRNA agent are modified. For example, if 50% of all nucleotides of a double-stranded iRNA agent are modified, then 50% of all nucleotides of the double-stranded iRNA agent include a modification as described herein.

[0042] In one embodiment, at least 50% of the nucleotides of the double-stranded iRNA agent are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-deoxy, or 2'-fluoro.

[0043] In one embodiment, at least 50% of the nucleotides of the antisense are independently modified with LNA, CeNA, 2'-methoxyethyl, or 2'-deoxy.

[0044] In some embodiments, a double-stranded iRNA agent includes fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, or fewer than 3 2'-F modifications. In some embodiments, a double-stranded iRNA agent has fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 2'-F modifications on the sense strand. In some embodiments, a double-stranded iRNA agent has fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, fewer than 2 2'-F modifications on the antisense strand.

[0045] In some embodiments, the double-stranded iRNA agent has one or more 2'-F modifications anywhere in the sense or antisense strand.

[0046] In some embodiments, the sense strand comprises at least four 2'-F modifications, for example, at positions 7 and 9-11 counting from the 5' end of the sense strand.

[0047] In some embodiments, the antisense strand comprises at least four 2'-F modifications, e.g., at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises at least six 2'-F modifications, e.g., at positions 2, 6, 8-9, 14, and 16, counting from the 5' end of the antisense strand.

[0048] In some embodiments, the double-stranded iRNA agent has less than 20%, less than 15%, less than 10%, less than 5%, or is substantially free of non-natural nucleotides. Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (2'-modified L-nucleosides, such as 2'-deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic, and open-chain alkyls.

[0049] In some embodiments, a double-stranded iRNA agent has more than 80%, more than 85%, more than 90%, more than 95%, or substantially 100% naturally occurring nucleotides. For the purposes of these embodiments, naturally occurring nucleotides can include those with 2'-OH, 2'-deoxy, and 2'-OMe.

[0050] In one embodiment, the double-stranded iRNA agent has sense and antisense strands each having a length of 15-30 nucleotides; and includes at least two consecutive phosphorothioate internucleotide linkages within positions 18-23 on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); and wherein the double-stranded iRNA agent has less than 20%, less than 15%, less than 10%, less than 5% natural nucleotides, or is substantially free of non-natural nucleotides.

[0051] In one embodiment, the double-stranded iRNA agent has sense and antisense strands each having a length of 15-30 nucleotides; and includes at least two consecutive phosphorothioate internucleotide linkages within positions 18-23 on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); and wherein the double-stranded iRNA agent has more than 80%, more than 85%, more than 95%, or substantially 100% natural nucleotides, e.g., those having 2'-OH, 2'-deoxy, or 2'-OMe.

[0052] In some embodiments, the penetration enhancer is selected from the group consisting of a fatty acid or a pharmaceutically acceptable salt thereof, a fatty acid derivative or a pharmaceutically acceptable salt thereof, a bile acid or a pharmaceutically acceptable salt thereof, a chelating agent, a surfactant, a non-chelating non-surfactant, and chitosan or a derivative thereof.

[0053] In some embodiments, the penetration enhancer is selected from the group consisting of arachidonic acid, oleic acid, lauric acid, capric acid, caprylic acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, C 1~10 The fatty acid or a pharmaceutically acceptable salt thereof is selected from the group consisting of alkyl esters, monoglycerides, diglycerides, and pharmaceutically acceptable salts thereof.

[0054] In some embodiments, the penetration enhancer is caprylic acid (C8), capric acid (C10), lauric acid (C12), oleic acid (C18), or a pharmaceutically acceptable salt thereof, e.g., the sodium salt of the above fatty acids.

[0055] In one embodiment, the penetration enhancer is sulcaprozate sodium.

[0056] In one embodiment, the penetration enhancer is chitosan or trimethylchitosan chloride.

[0057] In some embodiments, the oral formulation is configured for delivery as a capsule, a soft elastic gelatin capsule, a hard gelatin capsule, a caplet, an aerosol, a spray, a solution, a suspension, or an emulsion.

[0058] Another aspect of the invention relates to a method for reducing expression of a target gene in a subject, the method comprising orally administering to a subject in need thereof a formulation comprising: a) a double-stranded iRNA agent; and b) a penetration enhancer. The double-stranded iRNA agent comprises an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and, optionally, a carbohydrate-based ligand conjugated to at least one strand via a linker or carrier. The double-stranded iRNA agent also comprises 2'-OMe modifications to more than 15, more than 20, more than 25, or more than 30 nucleotides.

[0059] In some embodiments, the unit dose of the double-stranded iRNA agent is administered at about 50 mg or less per kg of body weight, e.g., about 40 mg or less per kg, about 30 mg or less per kg, about 25 mg or less per kg, about 20 mg or less per kg, about 15 mg or less per kg, about 10 mg or less per kg, about 5 mg or less per kg, about 3 mg or less per kg, about 2 mg or less per kg, about 1 mg or less per kg, about 0.5 mg or less per kg, or about 0.1 mg or less per kg of body weight. In some embodiments, the unit dose of the double-stranded iRNA agent is administered at about 1 to about 30 mg / kg of body weight, e.g., about 3 to about 25 mg / kg of body weight. In one embodiment, the dosage is calculated according to the oral bioavailability of the individual oligomer to obtain a dosage that allows for the maintenance of an effective concentration of the oligomer in the target tissue.

[0060] In some embodiments, the concentration of the penetration enhancer in the formulation is about 200 mM or less, e.g., about 150 mM or less, about 100 mM or less, about 80 mM or less, about 60 mM or less, about 50 mM or less, about 45 mM or less, or about 40 mM or less, about 35 mM or less, or about 30 mM or less.

[0061] In some embodiments, the formulation is configured for delivery as a capsule, a soft elastic gelatin capsule, a hard gelatin capsule, a caplet, an aerosol, a spray, a solution, a suspension, or an emulsion.

[0062] The above-described embodiments relating to double-stranded iRNA agents and their chemical modifications, including their conjugation to carbohydrate-based ligands and double-stranded iRNA agents, and penetration enhancers in the first aspect of the invention relating to oral formulations for reducing or inhibiting expression of a target gene in a subject are all suitable for this aspect of the invention relating to methods of reducing expression of a target gene in a subject. [Brief explanation of the drawings]

[0063] [Figure 1] 1 is a graph showing relative F12 levels after oral delivery to fasted mice of doses of a formulation containing GalNAc-siRNA of 3 mg / kg (with 45.4 mM C10 in solution), 10 mg / kg (with 150 mM C10), and 25 mg / kg (with 37.5 mM C10), respectively, on days 0, 2, and 5. Comparisons were made with the same siRNA administered subcutaneously at a single dose of 0.75 mg / kg. [Figure 2] 1 is a graph showing relative F12 levels after oral delivery to fasted mice of a 25 mg / kg dose of a formulation containing GalNAc-siRNA (with 37.5 mM C10) on days 0, 2, and 5, compared to relative F12 levels after oral delivery to fasted mice of a 25 mg / kg dose of the same siRNA (without C10) on days 0, 2, and 5. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.75 mg / kg. [Figure 3]1 is a graph showing relative F12 levels after oral delivery to fasted mice of a 25 mg / kg dose of a formulation containing GalNAc-siRNA (with 37.5 mM C10) compared to relative F12 levels after oral delivery to non-fasted mice of a 25 mg / kg dose of the same siRNA (with 37.5 mM C10) on days 0, 2, and 5. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.75 mg / kg. [Figure 4] 1 is a graph showing relative F12 levels after oral delivery to fasted mice of a 25 mg / kg dose of a formulation containing GalNAc-conjugated siRNA (with 37.5 mM C10) on days 0, 2, and 5, compared to relative F12 levels after oral delivery to fasted mice of a 25 mg / kg dose of a formulation containing unconjugated siRNA (with 37.5 mM C10) on days 0, 2, and 5. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.75 mg / kg. [Figure 5] 12 is a graph showing relative F12 levels after oral delivery to fasted mice of a 25 mg / kg dose of a formulation containing GalNAc-siRNA (with 37.5 mM C10) on days 0, 2, and 5, compared to relative F12 levels after oral delivery to fasted mice of a 25 mg / kg dose of the same siRNA (with 37.5 mM C10) on days 0, 8, and 14. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.75 mg / kg. [Figure 6]1 is a graph showing relative plasma F12 levels after oral delivery to fasted mice of a 10 mg / kg dose of a formulation containing GalNAc-siRNA (with 150 mM C10) compared to relative plasma F12 levels after oral delivery to fasted mice of a 10 mg / kg dose of the same siRNA without GalNAc conjugation (with 150 mM C10, sodium caproate) on days 0, 2, and 5. Oral administration of PBS and subcutaneous administration of a single 0.75 mg / kg dose of the same GalNAc-siRNA were used as comparisons. [Figure 7] 10 is a graph showing relative plasma F12 levels after oral delivery of a single 30 mg / kg dose of a formulation containing GalNAc-siRNA (with 150 mM C10, sodium caproate) to fasted or freely fed wild-type C57 / BL6 mice compared to relative plasma F12 levels after oral delivery of a single 30 mg / kg dose of the same GalNAc-siRNA (with 150 mM C10) to fasted or freely fed asialoglycoprotein receptor (ASGR) knockout (KO) mice. [Figure 8] 12 is a graph showing relative plasma F12 levels after oral delivery to fasted mice of single doses of 1, 3, and 10 mg / kg of a formulation containing GalNAc-siRNA (with 150 mM C10, sodium caproate), compared to relative plasma F12 levels after oral delivery to fasted mice of doses of the same GalNAc-siRNA (with 150 mM C10) at 1, 3, and 10 mg / kg, respectively, on days 0, 2, and 5. An oral formulation containing the same GalNAc-siRNA but without the vinylphosphonate (VP) at the 5' end of the antisense sequence was also used and administered to fasted mice at 3 mg / kg (with 150 mM C10) on days 0, 2, and 5. [Figure 9]12 is a graph showing relative plasma F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of a formulation containing GalNAc-siRNA (with 150 mM C10) on days 0, 2, and 5; relative plasma F12 levels after oral delivery to freely fed mice of a 3 mg / kg dose of the same formulation containing GalNAc-siRNA (with 150 mM C10) every 4 hours on day 0; and relative plasma F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of the same formulation containing GalNAc-siRNA (with 150 mM C10, sodium caproate) on days 0, 1, and 2. In all groups, formulations containing GalNAc-siRNA were orally administered in three doses at the same dosage level. [Figure 10] 1 is a graph showing relative F12 levels after oral delivery to NHPs of a formulation containing GalNAc-siRNA (with 150 mM C10 in solution). [Figure 11] Graph showing relative plasma F12 levels after oral delivery of a dose of a formulation containing 10 mg / kg GalNAc-siRNA (with 150 mM C10) to cynomolgus monkeys (N=4) on days 0, 2, and 5. [Figure 12] Figure 12 is a graph showing relative plasma TTR levels after oral delivery to cynomolgus monkeys of doses of a formulation containing GalNAc-siRNA (AD-157687 with 150 mM C10) at 3 mg / kg and 10 mg / kg, respectively, on days 0, 2, and 5. A comparison was made with the same GalNAc-siRNA administered subcutaneously at a single dose of 1 mg / kg. [Figure 13] 1 is a graph showing relative plasma TTR levels after oral delivery to cynomolgus monkeys of doses of a formulation containing GalNAc-siRNA (AD-87404 with 150 mM C10) at 3, 10, and 30 mg / kg, respectively, on days 0, 2, and 5. A comparison was made with the same GalNAc-siRNA administered subcutaneously at a single dose of 3 mg / kg. [Figure 14]

[0023] Figure 10 is a graph showing the amount of siRNA in plasma after oral delivery to cynomolgus monkeys of a single dose of 3 and 10 mg / kg of a formulation containing GalNAc-siRNA (AD-157687, together with 150 mM C10). A comparison was made with the same GalNAc-siRNA administered subcutaneously at a single dose of 1 mg / kg. [Figure 15]

[0023] Figure 12 is a graph showing the amount of siRNA in plasma after oral delivery to cynomolgus monkeys of a single dose of 3, 10, and 30 mg / kg of a formulation containing GalNAc-siRNA (AD-87404, together with 150 mM C10). A comparison was made with the same GalNAc-siRNA administered subcutaneously at a single dose of 3 mg / kg. [Figure 16]

[0023] Figure 12 is a graph showing the amount of siRNA in the liver after oral delivery to cynomolgus monkeys of doses of formulations containing 3 and 10 mg / kg GalNAc-siRNA (AD-157687, together with 150 mM C10), respectively. A comparison was made with the same GalNAc-siRNA administered subcutaneously at a single dose of 1 mg / kg. [Figure 17] 1 is a graph showing the amount of siRNA in the liver after oral delivery to cynomolgus monkeys of doses of a formulation containing 3, 10, and 30 mg / kg GalNAc-siRNA (AD-87404, together with 150 mM C10), respectively, on days 0, 2, and 5. A comparison was made with the same GalNAc-siRNA administered subcutaneously at a single dose of 3 mg / kg. [Figure 18]Relative F12 levels after oral delivery to fasted mice of a dose of a formulation containing 3 mg / kg GalNAc-siRNA (with 150 mM or 75 mM C10) on days 0, 2, and 5; relative F12 levels after oral delivery to fasted mice of a dose of a formulation containing 3 mg / kg of the same siRNA (with 150 mM or 75 mM C8) on days 0, 2, and 5; relative F12 levels after oral delivery to fasted mice of a dose of a formulation containing 3 mg / kg of the same siRNA (with 150 mM or 75 mM C12) on days 0, 2, and 5. Graph showing the relative F12 levels after oral delivery to fasted mice of a dose of a formulation containing the same siRNA (with 150 mM or 75 mM C18:1) at 3 mg / kg on days 0, 2, and 5; and the relative F12 levels after oral delivery to fasted mice of a dose of a formulation containing the same siRNA (with 75 mM C10 in combination with 75 mM C8) at 3 mg / kg on days 0, 2, and 5. Comparison was made using the same siRNA subcutaneously administered at a single dose of 0.15 mg / kg. [Figure 19] 1 is a graph showing relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of a formulation containing GalNAc-siRNA (with 150 mM or 75 mM C10) on days 0, 2, and 5, compared to relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of the same siRNA (with 150 mM or 75 mM C8) on days 0, 2, and 5. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.15 mg / kg. [Figure 20] 1 is a graph showing relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of a formulation containing GalNAc-siRNA (with 150 mM or 75 mM C10) on days 0, 2, and 5, compared to relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of the same siRNA (with 150 mM or 75 mM C12) on days 0, 2, and 5. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.15 mg / kg. [Figure 21] 1 is a graph showing relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of a formulation containing GalNAc-siRNA (with 150 mM or 75 mM C10) on days 0, 2, and 5, compared to relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of the same siRNA (with 150 mM or 75 mM C18:1) on days 0, 2, and 5. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.15 mg / kg. [Figure 22] 12 is a graph showing relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of a formulation containing GalNAc-siRNA (with 150 mM or 75 mM C10) on days 0, 2, and 5, compared to relative F12 levels after oral delivery to fasted mice of a 3 mg / kg dose of the same siRNA (with 75 mM C10 in combination with 75 mM C8) on days 0, 2, and 5. A similar comparison was made using the same siRNA administered subcutaneously at a single dose of 0.15 mg / kg. [Figure 23] 1 is a graph showing relative plasma F12 levels after oral delivery to fasted mice of doses of formulations containing GalNAc-siRNA with 75 mM of various penetration enhancers, including sodium caproate (C10), salcaprozate sodium (SNAC), ethylenediaminetetraacetic acid (EDTA), sodium oleate (C18:1), sodium laurate (C12), and sodium caprylate (C8), each at 3 mg / kg, on days 0, 2, and 5. Comparisons were made using the same GalNAc-siRNA administered subcutaneously at a single dose of 0.15 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0064] In particular, the inventors have found that the formulations described herein provide unexpectedly good and robust results for in vivo oral delivery of double-stranded iRNA agents, achieving effective and efficient oral delivery of double-stranded iRNA agents in clinically relevant dosing regimens.

[0065] One aspect of the invention provides an oral formulation for reducing or inhibiting expression of a target gene in a subject. The oral formulation includes a) a double-stranded iRNA agent and b) a penetration enhancer. The double-stranded iRNA agent includes an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; and a carbohydrate-based ligand conjugated to at least one of the strands, optionally via a linker or carrier. The double-stranded iRNA agent also includes 2'-OMe modifications to more than 15, more than 20, more than 25, or more than 30 nucleotides.

[0066] Ligand The double-stranded iRNA agents of the invention are further modified by the covalent attachment of one or more targeting ligands, such as carbohydrate-based ligands.

[0067] As used herein, the term "targeting ligand" refers to any molecule that provides increased affinity for a selected target, e.g., a cell, cell type, tissue, organ, body region, or compartment, e.g., a cell, tissue, or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0068] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3 (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent fucose, glycosylated polyamino acids, and lectins. The term multivalent indicates the presence of two or more monosaccharide units. Such monosaccharide subunits can be linked to each other via glycosidic bonds or to a backbone molecule.

[0069] As ligands, several folates and folate analogs suitable for the present invention are described in U.S. Pat. Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, the entire contents of which are incorporated herein by reference.

[0070] When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties while other ligands have different properties. For example, the ligands may have targeting properties, endosomolytic activity, or PK modulating properties. In a preferred embodiment, the ligands all have different properties.

[0071] As used herein, the terms "PK-modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of the compositions of the present invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binders, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, transthyretin binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Oligomeric compounds containing several phosphorothioate intersugar linkages are also known to bind to serum proteins. Therefore, short oligomeric compounds, e.g., oligonucleotides containing about 5-30 nucleotides (e.g., 5-25 nucleotides, preferably 5-20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. PK-modulating oligonucleotides can contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in a PK-modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for the present invention as PK-modulating ligands. Binding to serum components (e.g., serum proteins) can be predicted from albumin binding assays such as those described in Oravcova, et al., Journal of Chromatography B (1996), 677:1-27.

[0072] Generally, conjugate groups modulate one or more properties of the attached double-stranded iRNA agent, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are routinely used in chemistry and are linked to a parent compound, such as an oligomeric compound, either directly or via an optional linking moiety or group.

[0073] A preferred list of conjugate groups includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folates, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, and dyes.

[0074] Preferred conjugate groups suitable for the present invention include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); aliphatic chains, e.g., 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); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0075] In general, a wide variety of entities, e.g., ligands, can be attached to the oligomeric compounds described herein. Ligands can include naturally occurring molecules or recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, 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, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic group, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimeric polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, thyrotropin, melanotropin , lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralens, mitomycin C), porphyrins (e.g., TPPC4, Texas phyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, 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)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., α-helical peptides, amphipathic peptides, RGD peptides, cell-penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen, aspirin, vitamin E, folic acid ), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharides, p38 These include activators of MAP kinase, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor α (TNFα), interleukin-1β, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell-penetrating agents (e.g., alpha-helical cell-penetrating agents).

[0076] Peptide and peptidomimetic ligands include natural or modified peptides, e.g., D- or L-peptides; α-, β-, or γ-peptides; N-methylpeptides; azapeptides; peptides with one or more amide bonds, i.e., peptides, one or more urea, thiourea, carbamate, or sulfonylurea bonds substituted; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands 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.

[0077] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, Esculentinis-1, and caerin.

[0078] As used herein, the term "endosomolytic ligand" refers to a molecule that has endosomolytic properties. An endosomolytic ligand promotes lysis of a composition of the invention, or a component thereof, and / or transport of a composition of the invention, or a component thereof, from a cellular compartment, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular endoplasmic reticulum, to the cytoplasm of a cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEI), linear or branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charge, dendrimers with masked or unmasked cationic or anionic charge, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0079] Exemplary endosomolytic / fusogenic peptides include, but are not limited to, AALEALAEALAEALAEALEALAEAAAAGGC (GALA) (SEQ ID NO: 1); AALEALAEALAEALAEALAEALAEALAAAAGGC (EALA) (SEQ ID NO: 2); ALEALAEALEALEALAEA (SEQ ID NO: 3); GLFEAIEGFIENGWEGMIWDYG (INF-7) (SEQ ID NO: 4); GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2) (SEQ ID NO: 5); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMIDGWYGC (diINF-7) (SEQ ID NO: 6); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3) (SEQ ID NO: 7); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF) (SEQ ID NO: 8); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3) (SEQ ID NO: 9); GLFEAIEGFIENGWEGnIDGKGLFEAIEGFIENGWEGnIDG (INF-5, n is norleucine) (SEQ ID NO: 10); LFEALLELLESLWELLLEA (JTS-1) (SEQ ID NO: 11); GLFKALLKLLKSLWKLLLKA (ppTG1) (SEQ ID NO: 12); GLFRALLRLLRSLWRLLLRA (ppTG20) (SEQ ID NO: 13); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA) (SEQ ID NO: 14); GLFFEAIAEFIEGGWEGLIEGC (HA) (SEQ ID NO: 15); GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin) (SEQ ID NO: 16); H5WYG (SEQ ID NO: 17); and CHK6HC (SEQ ID NO: 18).

[0080] Without wishing to be bound by theory, fusogenic lipids fuse with membranes, thereby destabilizing them. Fusogenic lipids typically have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0081] Synthetic polymers with endosomolytic activity suitable for the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410; 2009 / 0023890; 2008 / 0287630; 2008 / 0287628; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, the entire contents of which are incorporated herein by reference.

[0082] Exemplary cell-penetrating peptides include, but are not limited to, RQIKIWFQNRRMKWKK (penetratin) (SEQ ID NO: 19); GRKKRRQRRRPPQC (Tat fragment 48-60) (SEQ ID NO: 20); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence-based peptide) (SEQ ID NO: 21); LLIILRRRIRKQAHAHSK (PVEC) (SEQ ID NO: 22); GWTLNSAGYLLKINLKALAALAKKIL (transportan) (SEQ ID NO: 23); KLALKLALKALKAALKLA (amphipathic model peptide) (SEQ ID NO: 24); RRRRRRRRR (Arg9) (SEQ ID NO: 25); KFFKFFKFFK (bacterial cell wall-penetrating peptide) (SEQ ID NO: 26); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37) (SEQ ID NO: 27); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO: 28); ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO: 29); DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO: 30); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID NO: 31); ILPWKWPWWPWRR-NH2 (indolicidin) (SEQ ID NO: 32); AAVALLPAVLLALLAP (RFGF) (SEQ ID NO: 33); AALLPVLLAAP (RFGF analog) (SEQ ID NO: 34); and RKCRIVVIRVCR (bactenecin) (SEQ ID NO: 35).

[0083] Exemplary cationic groups include, but are not limited to, O-AMINE (AMINE = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, e.g., O(CH) nAMINE, (e.g., AMINE = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH(CHCHNH) n Included are protonated amino groups derived from CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0084] A ligand or linking ligand can be present on a monomer when the monomer is incorporated into a component of a double-stranded iRNA agent of the invention (e.g., a double-stranded iRNA agent or linker of the invention). In some embodiments, a ligand can be incorporated into a "precursor" monomer by coupling after the monomer has been incorporated into a component of a double-stranded iRNA agent of the invention (e.g., a double-stranded iRNA agent or linker of the invention). For example, a monomer with an amino-terminal tether (i.e., no ligand attached), e.g., monomer-linker-NH, can be incorporated into a component of a compound of the invention (e.g., a double-stranded iRNA agent or linker of the invention). In a subsequent operation, i.e., after incorporation of the precursor monomer into a component of a compound of the invention (e.g., a double-stranded iRNA agent or linker of the invention), a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the tether of the precursor monomer.

[0085] In another example, monomers bearing chemical groups suitable for participating in click chemistry reactions can be incorporated into tethers / linkers, for example, azide- or alkyne-terminated tethers / linkers. Subsequent to this, i.e., after the precursor monomers are incorporated into the chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer by linking the alkyne and azide together.

[0086] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of the double-stranded iRNA agent of the present invention. Conjugation to a purine nucleobase or a derivative thereof can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is bound to the conjugate moiety. Conjugation to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be substituted with the conjugate moiety. When a ligand is conjugated to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions required for base pairing.

[0087] Conjugation to the sugar moiety of the nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached to the conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as an abasic residue. The internucleoside linkage can also carry a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0088] There are many methods for preparing the conjugate of oligonucleotide.Generally, oligonucleotide is linked to conjugate moiety by contacting the reactive group (for example, OH, SH, amine, carboxyl, aldehyde, etc.) of oligonucleotide with the reactive group of conjugate moiety.In some embodiments, one reactive group is electrophilic, and the other is nucleophilic.

[0089] For example, the electrophilic group can be a carbonyl-containing functional group, and the nucleophilic group can be an amine or thiol. Methods for conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in literature, such as, for example, Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, the entire contents of which are incorporated herein by reference.

[0090] Ligands can be attached to double-stranded iRNA agents of the invention via linkers or carrier monomers, e.g., ligand carriers. Carriers include (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond available and suitable for incorporation of a carrier monomer into the backbone of an oligonucleotide, e.g., a phosphate backbone, or, e.g., a sulfur-containing modified phosphate backbone. A "tether attachment point" (TAP) refers to an atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of a carrier monomer that connects a selected moiety. The selected moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the carrier monomer by an intervening tether. Thus, carriers often include functional groups, e.g., amino groups, or generally allow for a bond suitable for incorporation or linking of another chemical entity, e.g., a ligand, to a constituent atom.

[0091] Representative United States patents that teach the preparation of nucleic acid conjugates include, but are not limited to, U.S. Pat. Nos. 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; 5,580,731; 5,591,584 ... Specification No. 5,109,124; Specification No. 5,118,802; Specification No. 5,138,045; Specification No. 5,414,077; Specification No. 5,486,603; Specification No. 5,512,439; Specification No. 5,578,718; Specification No. 5,608,046 ; Specification No. 4,587,044; Specification No. 4,605,735; Specification No. 4,667,025; Specification No. 4,762,779 Specifications; Specification No. 4,789,737; Specification No. 4,824,941; Specification No. 4,835,263; Specification No. 4,876,335 Specification; Specification No. 4,904,582; Specification No. 4,958,013; Specification No. 5,082,830; Specification No. 5,112,96 Specification No. 3; Specification No. 5,214,136; Specification No. 5,082,830; Specification No. 5,112,963; Specification No. 5,149,7 Specification No. 82; Specification No. 5,214,136; Specification No. 5,245,022; Specification No. 5,254,469; Specification No. 5,258 ,506 specification; 5,262,536 specification; 5,272,250 specification; 5,292,873 specification; 5,31 Specification No. 7,098; Specification No. 5,371,241, Specification No. 5,391,723; Specification No. 5,416,203, Specification No. 5, Specification No. 451,463; Specification No. 5,510,475; Specification No. 5,512,667; Specification No. 5,514,785; Specification No. 5 ,565,552; 5,567,810; 5,574,142; 5,585,481; Specification No. 5,587,371; Specification No. 5,595,726; Specification No. 5,597,696; Specification No. 5,599,923;Nos. 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; and 6,559,279.

[0092] In some embodiments, a double-stranded iRNA agent can further include one or more other ligands, such as a lipophilic moiety, conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier. The terms "lipophilic portion" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One method for characterizing the lipophilicity of a lipophilic moiety is the octanol-water partition coefficient, logK ow where K ow is the ratio of a chemical's concentration in the octanol phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to the chemical's components calculated using first-principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment over water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its log K ow is greater than 0. Typically, a lipophilic moiety has a log K of greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow For example, the log K of 6-aminohexanol owFor example, the log K of cholesteryl N-(hexan-6-ol) carbamate is predicted to be about 0.7. ow is predicted to be 10.7.

[0093] The lipophilicity of a molecule can be altered with respect to the functional groups it possesses. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value may be increased or decreased.

[0094] Alternatively, the hydrophobicity of the double-stranded iRNA agent conjugated with one or more lipophilic moieties can be measured by its protein binding properties.For example, the unbound fraction in the plasma protein binding assay of double-stranded iRNA agent can be determined to be correlated with the relative hydrophobicity of double-stranded iRNA agent, which can be positively correlated with the silencing activity of double-stranded iRNA agent.

[0095] In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the double-stranded iRNA agent, as measured by the fraction of unbound siRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for improved in vivo delivery of siRNA.

[0096] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded iRNA agent provides optimal hydrophobicity for improved in vivo delivery of the siRNA.

[0097] In certain embodiments, the lipophilic moiety is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polyalicyclic, compounds such as steroids (e.g., sterols) or straight- or branched-chain aliphatic hydrocarbons. The lipophilic moiety generally comprises a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, e.g., oxygen or nitrogen atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C6 30 Hydrocarbons (e.g., C6-C 18 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpene, C 15 Sesquiterpene, C 20 Diterpenes, C 30 Triterpenes and C 40 tetraterpenes), and other polyalicyclic hydrocarbons. For example, the lipophilic moiety may be C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 In some embodiments, the lipophilic moiety may contain a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., straight chain C6-C 18 In one embodiment, the lipophilic moiety contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C 16 alkyl or alkenyl).

[0098] The lipophilic moiety can be attached to the iRNA agent by any method known in the art, such as through a functional group already present in the lipophilic moiety or introduced into the iRNA agent, such as a hydroxy group (e.g., -CO-CH-OH). Functional groups already present in the lipophilic moiety or introduced into the iRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0099] Conjugation of the iRNA agent and the lipophilic moiety can occur, for example, by formation of an ether or carboxyl or carbamoyl ester bond between a hydroxy and alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chain or branched-chain; and saturated or unsaturated). The alkyl group R can be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group, etc.

[0100] In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that includes an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0101] In another embodiment, the lipophilic moiety is a steroid, such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. "Cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition, or removal of substituents.

[0102] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" refers broadly to monocyclic and polycyclic aromatic hydrocarbons. Aromatic groups include, but are not limited to, C6-C8 aromatic groups containing 1-3 aromatic rings, which may be optionally substituted. 14"Aralkyl" or "arylalkyl" groups comprising an aryl group covalently bonded to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and "heteroaryl" groups. As used herein, the term "heteroaryl" refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; 6, 10, or 14 pi-electrons shared in a cyclic arrangement; and, in addition to carbon atoms, 1 to about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0103] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group has 1 to about 4, preferably 1 to about 3, and more preferably 1 or 2 non-hydrogen substituents. Suitable substituents include, but are not limited to, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0104] In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety binds to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, immunoglobulins, lipoproteins, α-2-macroglobulin, or α-1-glycoprotein.

[0105] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Procedures for the synthesis of naproxen can be found in U.S. Pat. Nos. 3,904,682 and 4,009,197, the entire contents of which are incorporated herein by reference. Naproxen has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid and the structure: [ka] is.

[0106] In certain embodiments, the ligand is ibuprofen or an ibuprofen structural derivative. Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No. 3,228,831, the entire contents of which are incorporated herein by reference. The structure of ibuprofen is: [ka] is.

[0107] Further exemplary aralkyl groups are set forth in US Pat. No. 7,626,014, the entire contents of which are incorporated herein by reference.

[0108] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0109] In some embodiments, the lipophilic moiety is a C-C 30Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexanoic acid, vitamin A, vitamin E, cholesterol, etc.) or C6 to C 30 Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0110] In certain embodiments, two or more lipophilic moieties can be incorporated into a double-stranded iRNA agent, particularly if the lipophilic moieties have low lipophilicity or hydrophobicity. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of a double-stranded iRNA agent. In one embodiment, one or more lipophilic moieties are incorporated into each strand of a double-stranded iRNA agent. In one embodiment, two or more lipophilic moieties are incorporated into the same position of a double-stranded iRNA agent (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage). This can be accomplished, for example, by conjugating two or more lipophilic moieties via a carrier, and / or by conjugating two or more lipophilic moieties via a branched linker, and / or by conjugating two or more lipophilic moieties via one or more linkers, with one or more linkers connecting the lipophilic moieties consecutively.

[0111] The lipophilic moiety can be conjugated to the iRNA agent via a direct bond to the iRNA agent's ribosugar. Alternatively, the lipophilic moiety can be conjugated to the double-stranded iRNA agent via a linker or carrier.

[0112] In certain embodiments, the ligand can be conjugated to the iRNA agent via one or more linkers (tethers).

[0113] In one embodiment, the ligand is conjugated to the double-stranded iRNA agent via a linker that contains an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0114] Linker / Tether The linker / tether is connected to the ligand at a "tethering attachment point (TAP)". The linker / tether can be any C1-C 100 Carbon-containing moieties (e.g., C1-C 75 , C1~C 50 , C1~C 20 , C1~C 10 ;C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 ) and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) group in the linker / tether that can serve as a point of attachment for a ligand. Non-limiting examples of linkers / tethers (underlined) include: [ka] wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R"" is a C1-C6 alkyl. Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or a hydrazino group, -NHNH2. The linker / tether may be optionally substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or may optionally contain one or more additional heteroatoms, e.g., N, O, or S. Preferred linking ligands include, for example, [ka] In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can be acylated, for example, with C(O)CF.

[0115] In some embodiments, the linker / tether may terminate in a mercapto group (i.e., SH) or an olefin (e.g., CH=CH). For example, the tether may be: [ka] where n can be as described elsewhere. The tether can be optionally substituted, for example, with hydroxy, alkoxy, perhaloalkyl, and / or can optionally contain one or more additional heteroatoms, for example, N, O, or S. The double bond can be cis or trans or E or Z.

[0116] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at the terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, an aldehyde, an alkyl halide, a mesylate, a tosylate, a nosylate, or a brosylate, or an activated carboxylic acid ester, such as an NHS ester, or a pentafluorophenyl ester. Preferred linkers / tethers (underlined) include: [ka] where n is 1 to 6 and R"" is C1 to C6 alkyl; or [ka] where n is 1 to 6 and R'''' is C1 to C6 alkyl; [ka] where n is 1 to 11 and R"" is C1 to C6 alkyl; or [ka] where n can be as described elsewhere and R"" is a C1-C6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be performed by coupling a nucleophilic group, e.g., a thiol or amino group, of the ligand with an electrophilic group on the tether.

[0117] In other embodiments, it may be desirable for the monomer to contain a phthalimide group (K) at the terminal position of the linker / tether. [ka]

[0118] In other embodiments, other protected amino groups can be at the terminal positions of the linker / tether, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety can be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0119] Any of the linkers / tethers described herein may contain one or more additional linking groups, such as -O-(CH) n -, -(CH2) n -SS-, -(CH2) n It may further comprise - or -(CH=CH)-.

[0120] Cleavable Linker / Tether In some embodiments, at least one of the linkers / tethers can be a redox-cleavable linker, an acidic-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0121] In one embodiment, at least one of the linkers / tethers can be a reductively cleavable linker (eg, a disulfide group).

[0122] In one embodiment, at least one of the linkers / tethers can be an acid-cleavable linker (eg, a hydrazone group, an ester group, an acetal group, or a ketal group).

[0123] In one embodiment, at least one of the linkers / tethers can be an esterase-cleavable linker (eg, an ester group).

[0124] In one embodiment, at least one of the linkers / tethers can be a phosphatase-cleavable linker (eg, a phosphate group).

[0125] In one embodiment, at least one of the linkers / tethers can be a peptidase-cleavable linker (eg, a peptide bond).

[0126] Cleavable linkers are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or present at higher levels or activity within cells than in serum or blood. Examples of such degrading agents include intracellular oxidases or reductases or reducing agents that can degrade redox-cleavable linkers by reduction, such as mercaptans, which are selective for specific substrates or have no substrate specificity; esterases; endosomes or agents that can create an acidic environment, such as a pH of 5 or less; enzymes that can hydrolyze or degrade acidic cleavable linkers by functioning as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0127] Cleavable linking groups, e.g., disulfide bonds, are sensitive to pH. The pH of human serum is 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 tethers have linking groups that are cleaved at a preferred pH, thereby releasing the iRNA agent from the ligand (e.g., a targeting or cell-permeable ligand, e.g., cholesterol) into the cell or into a desired compartment of the cell.

[0128] The chemical bond (e.g., linking group) linking the ligand to the iRNA agent can include a disulfide bond. When the iRNA agent / ligand complex is taken up into a cell by endocytosis, the acidic environment of the endosome causes the disulfide bond to be cleaved, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. 19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002). The ligand can be a secondary therapeutic agent that can complement the therapeutic effect of the targeting ligand or iRNA agent.

[0129] The tether may contain a linking group that can be cleaved by a specific enzyme. The type of linking group incorporated into the tether may vary depending on the cell targeted by the iRNA agent. For example, an iRNA agent targeting mRNA in liver cells may be conjugated to a tether containing an ester group. Liver cells are rich in esterases, and therefore, the tether is cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Cleavage of the tether releases the iRNA agent from the ligand attached to the distal end of the tether, thereby potentially enhancing the silencing activity of the iRNA agent. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0130] Tethers that include peptide bonds can be conjugated to iRNA agents that target cell types rich in peptidases, such as hepatocytes and synovial cells. For example, iRNA agents targeted to synovial cells for the treatment of inflammatory diseases (e.g., rheumatoid arthritis) can be conjugated to tethers that include peptide bonds.

[0131] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degradation agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or in contact with other non-target tissues, such as tissues to which the iRNA agent is exposed when administered to a subject. Thus, the relative ease of cleavage can be determined between a first condition and a second condition, where the first condition is selected to indicate cleavage within target cells, and the second condition is selected to indicate cleavage in other tissues or body fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2-fold, 4-fold, 10-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).

[0132] Redox-cleavable linking groups One class of cleavable linkers is redox-cleavable linkers, which are cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (—SS—). To determine whether a candidate cleavable linker is a suitable “reductively cleavable linker,” or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be verified. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic cleavage rates that may be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2-fold, 4-fold, 10-fold, or 100-fold faster inside 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 a 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.

[0133] Phosphate-based cleavable linkers Phosphate-based linking groups are cleaved by agents that decompose or hydrolyze phosphate groups. An example of an agent that cleaves phosphate groups within a cell is an intracellular enzyme, such as a phosphatase. 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-, and -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-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0134] Acid cleavable linking group An acidic cleavable tether is a tether that is cleaved under acidic conditions. In a preferred embodiment, the acidic cleavable tether is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less) or by an agent that can function as a general acid, such as an enzymatic agent. In cells, certain organelles with low pH, such as endosomes and lysosomes, can provide a cleavage environment for the acidic cleavable tether. Examples of acidic cleavable tethers include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acidic cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) 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.

[0135] Ester-based linking group Ester-based linking groups are cleaved by intracellular enzymes, such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0136] Peptide-based cleavable linkers Peptide-based linking groups are cleaved by intracellular enzymes, such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linking groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and do not include the entire amide functionality. Peptide cleavable linking groups have the general formula -NHCHR 1 C(O)NHCHR 2 C(O)—, where R 1 and R 2 are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0137] Biocleavable linkers / tethers Linkers can also include biocleavable linkers, which are nucleotide and non-nucleotide linkers or combinations thereof, that link two parts of a molecule, for example, one or both strands of two individual siRNA molecules to generate bis(siRNA). In some embodiments, simple electrostatic or stacking interactions between two individual siRNAs can represent a linker. Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and their derivatives, aliphatic, alicyclic, heterocyclic, and combinations thereof.

[0138] In some embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0139] In one embodiment, the biocleavable carbohydrate linker can have 1-10 sugar units with at least one aromatic linkage capable of linking two siRNA units. When more than one sugar is present, the units can be linked via 1-3, 1-4, or 1-6 sugar linkages or via alkyl chains.

[0140] Exemplary biocleavable linkers include: [ka] [ka] [ka] Examples include:

[0141] Further description of biocleavable linkers can be found in PCT Application No. PCT / US18 / 14213, entitled "Endosomal Cleavable Linkers," filed January 18, 2018, the entire contents of which are incorporated herein by reference.

[0142] Carrier In certain embodiments, the ligand is conjugated to the iRNA agent via a carrier that substitutes one or more nucleotides.

[0143] The carrier may be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from the group consisting of cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol skeleton or a diethanolamine skeleton.

[0144] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the double-stranded iRNA agent.

[0145] In other embodiments, the carrier replaces a nucleotide at the end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide at the 3' end of the sense strand, thereby functioning as an end cap to protect the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine. For example, the carrier can be cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0146] Ribonucleotide subunits in which the ribose sugar of the subunit has been replaced in this manner are called ribose-replacement modified subunits (RRMS). The carrier may be a cyclic or acyclic moiety and comprises two "backbone attachment points" (e.g., hydroxyl groups) and a ligand (e.g., a carbohydrate-based ligand). The ligand may be directly attached to the carrier or indirectly attached to the carrier by an intervening linker / tether, as described above. [ka]

[0147] The ligand-conjugated monomer subunit can be the 5' or 3' terminal subunit of an iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit can occupy an internal position, and both "W" groups can be one or more unmodified or modified ribonucleotides. Two or more ligand-conjugated monomer subunits can be present in an iRNA agent.

[0148] Monomers based on sugar substitutions, e.g., ligand-conjugated monomers (cyclic) Monomers based on cyclic sugar substitutions, e.g., ligand-conjugate monomers based on the substitutions, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) shown below, in which the preferred backbone attachment points are R 1 or R 2 ;R 3 or R 4 ; or Y is CR 9 R 10 If R 9 and R 10 (two positions may be selected from two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 The preferred tether attachment point is R 7 ; When X is CH2, R 5 or R 6 Carriers are described below as substances that can be incorporated into the chain. Thus, the structure may have one (for terminal positions) or two (for internal positions) attachment points, e.g., R 1 or R 2 ;R 3 or R 4 ; or R 9 or R 10 (Y is CR 9 R 10 It is understood that the term "R" also encompasses cases where the R group is linked to a phosphate or modified phosphate, e.g., a sulfur-containing backbone. For example, one of the R groups listed above can be -CH-, where one bond is linked to the carrier and one is linked to a backbone atom, e.g., the linking oxygen or central phosphorus atom. [ka] During the ceremony: X is N(CO)R 7 , N.R. 7 or CH2; Y is NR 8 ,O,S,CR 9 R 10and; Z is CR 11 R 12 is or does not exist; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 each of which is independently H, OR a , or (CH2) n OR b where R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of the a and / or (CH2) n OR b and; R 5 , R 6 , R 11 , and R 12 each independently comprising a ligand, H, one to three R 13 optionally substituted C1-C6 alkyl, or C(O)NHR 7 or R 5 and R 11 Together, R 14 and optionally substituted C3-C8 cycloalkyl; R 7 can be a ligand, e.g., R 7 is R d or R 7 is a ligand, e.g., NR, that is indirectly linked to a carrier, e.g., via a tether moiety. c R d C1~C substituted with 20 Alkyl; or NHC(O)R d C1~C substituted with 20 may be alkyl; R 8 is H or C1-C6 alkyl; R 13 is hydroxy, C1-C4 alkoxy, or halo; R 14is NR c R 7 and; R 15 is cyano, optionally substituted C1-C6 alkyl, or C2-C6 alkenyl; R 16 is C1~C 10 is alkyl; R 17 is a liquid or solid phase carrier reagent; L is -C(O)(CH2) q C(O)- or -C(O)(CH2) q S- and; R a is a protecting group, such as CAr; (e.g., dimethoxytrityl group) or Si(X 5’ )(X 5” )(X 5”’ ), where (X 5’ ),(X 5” ), and (X 5”’ ) is as described above. R b is P(O)(O - )H, P(OR 15 )N(R 16 )2 or LR 17 and; R c is H or C1-C6 alkyl; R d is H or a ligand; Each Ar is independently selected from C1-C4 alkoxy, C6-C 10 is aryl; n is 1 to 4; q is 0 to 4.

[0149] Exemplary carriers include, for example, those in which X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is absent; or X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R12 or X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 or X is CH; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2) or indane ring system, for example, X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 taken together to form a C5 cycloalkyl (H, z=1).

[0150] In certain embodiments, the carrier may be based on a pyrroline ring system or a 4-hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z does not exist (D). [ka] OFG 1 is preferably a carbon atom in the five-membered ring (-CH2OFG in D) 1 ) is attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, which is linked to one of the OFG 2 is preferably a carbon atom in the five-membered ring (-OFG in D) 2 ) for pyrroline-based carriers, -CH2OFG 1 may be attached to C-2, OFG 2may be attached to C-3; or -CH2OFG 1 may be attached to C-3, OFG 2 may be attached to C-4. In certain embodiments, CH2OFG 1 and OFG 2 may be geminally substituted at one of the above carbons. For 3-hydroxyproline-based carriers, -CHOFG 1 may be attached to C-2, OFG 2 may be attached to C-4. Thus, pyrroline-based and 4-hydroxyproline-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, CHOFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the formula (I) and (II) both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration, or vice versa). The tether attachment point is preferably nitrogen. Preferred examples of carrier D include the following: [ka] Examples include:

[0151] In certain embodiments, the carrier may be based on a piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12is. [ka] OFG 1 is preferably a carbon atom in the six-membered ring [—(CH) in E] n OFG 1 OFG is bonded to a primary carbon, for example, an exocyclic alkylene group, for example, a methylene group (n=1) or an ethylene group (n=2), which is linked to one of the following: 2 is preferably a carbon atom in the six-membered ring (-OFG in E) 2 ) is directly bonded to one of the -(CH2) n OFG 1 and OFG 2 may be geminally positioned on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, -(CH) n OFG 1 and OFG 2 may be vicinal on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-3. Thus, a piperidine-based monomer may contain a bond (e.g., a carbon-carbon bond) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, -(CH2) n OFG 1 and OFG 2can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the R and S configurations may both have the R and S configurations, or one center may have the R and S configurations, or vice versa.) The tether attachment point is preferably nitrogen.

[0152] In certain embodiments, the carrier may be based on a piperazine ring system (F), e.g., X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or a morpholine ring system (G), for example, X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 is. [ka] OFG 1 is preferably a carbon atom in a six-membered ring (-CH2OFG in F or G) 1 ) is attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, which is linked to one of the OFG 2 is preferably a carbon atom in a six-membered ring (-OFG in F or G) 2 ) for both F and G. 1 may be attached to C-2, OFG 2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG 1 and OFG 2may be geminally substituted on one of the carbons. Thus, piperazine- and morpholine-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, CHOFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the R and S configurations may both have the R and S configurations; or one center may have the R and S configurations, or vice versa). R''' may be, for example, a C1-C6 alkyl, preferably CH3. The tether attachment points are preferably nitrogen in both F and G.

[0153] In certain embodiments, the carrier may be based on a decalin ring system, e.g., X is CH; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2) or indane ring system, e.g., X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C5 cycloalkyl (H, z=1). [ka] OFG 1is preferably —(CH) at C-2, C-3, C-4, or C-5 [H in —(CH) n OFG 1 ], for example, an exocyclic methylene group (n=1) or an ethylene group (n=2). OFG 2 is preferably at C-2, C-3, C-4, or C-5 (H in -OFG 2 ) is directly bonded to one of the -(CH2) n OFG 1 and OFG 2 may be geminally positioned on the ring, i.e., both groups may be attached to the same carbon, for example, at C-2, C-3, C-4, or C-5. Alternatively, -(CH) n OFG 1 and OFG 2 may be vicinal on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-5; or -(CH2) n OFG 1 may be attached to C-5, OFG 2 may be attached to C-4. Thus, decalin or indane-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, -(CH2)n OFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the formula (I) and (II) both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration, or vice versa.) In preferred embodiments, the substituents at C-1 and C-6 are trans relative to each other. The tether attachment point is preferably at C-6 or C-7.

[0154] Other carriers may include those based on 3-hydroxyproline (J). [ka] Therefore, -(CH2) n OFG 1 and OFG 2 can be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the R and S configurations may both have the R and S configurations, or one center may have the R and S configurations, or vice versa.) The tether attachment point is preferably nitrogen.

[0155] Details regarding more representative cyclic sugar-based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, the entire contents of which are incorporated herein by reference.

[0156] Monomers based on sugar substitution (acyclic) Acyclic sugar-substitution-based monomers, e.g., sugar-substitution-based ligand-conjugate monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers are represented by formula LCM-3 or LCM-4: [ka] may have:

[0157] In some embodiments, each of x, y, and z can be, independently of one another, 0, 1, 2, or 3. When y and z are different in formula LCM-3, the tertiary carbon can have either the R or S configuration. In a preferred embodiment, in formula LCM-3 (e.g., based on serinol), x is 0, and y and z are each 1; in formula LCM-3, y and z are each 1. Each of formulas LCM-3 or LCM-4 below can be optionally substituted, for example, with hydroxy, alkoxy, or perhaloalkyl.

[0158] Details regarding more representative acyclic sugar-based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, the entire contents of which are incorporated herein by reference.

[0159] In some embodiments, the double-stranded iRNA agent includes one or more ligands, such as carbohydrate-based ligands, conjugated to the 5'-end of the sense strand or the 5'-end of the antisense strand.

[0160] In certain embodiments, the ligand, such as a carbohydrate-based ligand, is conjugated to the 5' end of the chain via a carrier and / or a linker. In one embodiment, the ligand, such as a carbohydrate-based ligand, has the formula: [ka] R is a ligand, such as a carbohydrate-based ligand.

[0161] In some embodiments, the double-stranded iRNA agent includes one or more ligands, such as carbohydrate-based ligands, conjugated to the 3'-end of the sense strand or the 3'-end of the antisense strand.

[0162] In certain embodiments, the ligand, such as a carbohydrate-based ligand, is conjugated to the 3' end of the chain via a carrier and / or a linker. In one embodiment, the ligand, such as a carbohydrate-based ligand, has the formula: [ka] R is a ligand, such as a carbohydrate-based ligand.

[0163] In some embodiments, the targeting ligand targets liver tissue.In some embodiments, the targeting ligand is a carbohydrate-based ligand such as ASGPR ligand.In one embodiment, the targeting ligand is GalNAc conjugate.

[0164] In some embodiments, a targeting ligand, such as a carbohydrate-based ligand (eg, an ASGPR ligand), comprises one or more ligand moieties attached via a bivalent or trivalent branched linker.

[0165] In certain embodiments, the double-stranded iRNA agent comprises a targeting ligand having the structure shown below: [ka] Including, During the ceremony: L G is, independently for each occurrence, a ligand, e.g., a carbohydrate-based ligand, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, polysaccharide; Z', Z", Z"' and Z"" are each, independently for each occurrence, O or S.

[0166] In certain embodiments, the double-stranded iRNA agent has formula (II), (III), (IV), or (V): [ka] and a targeting ligand of During the ceremony: q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5C represents independently for each occurrence 0 to 20, and the repeat units may be the same or different; Q and Q' are, for each occurrence, either absent or -(P 7 -Q 7 -R 7 ) p -T 7 -or-T 7 -Q 7 -T 7’ -BT 8’ -Q 8 -T 8 and; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , P 7 , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C , T 7 , T 7’ , T 8 and T 8’each, independently for each occurrence, is absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; B is -CH2-N(B L )-CH2-; B L -T B -Q B -T B’ -R x and; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 7 , Q 8 and Q B is independently for each occurrence absent, alkylene, or substituted alkylene, and one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R'), C≡C or C(O); T B and T B’ each, independently for each occurrence, is absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH, CHNH, or CHO; R xis a lipophilic moiety (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)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazines); vitamins (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal); peptides; carbohydrate ligands, such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, polysaccharides; endosomolytic components, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-derivatized lithocholic acid), or cationic lipids; R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 are each independently 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 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C is, independently for each occurrence, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide; R' and R" are each independently H, C1-C6 alkyl, OH, SH, or N(R N )2; R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; R a is H or an amino acid side chain; Z', Z", Z"' and Z"" are each, independently for each occurrence, O or S; p is independent for each occurrence and ranges from 0 to 20.

[0167] As noted above, because a ligand can be conjugated to an iRNA agent via a linker or carrier, and because the linker or carrier can include a branched linker, an iRNA agent can include multiple ligands via the same or different backbone attachment points to the carrier or via a branched linker. For example, the branch point of a branched linker can be a divalent, trivalent, tetravalent, pentavalent, or hexavalent atom, or a group exhibiting such multivalency. In certain embodiments, the branch point is -N, -N(Q)-C, -OC, -SC, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)OC; where Q is, independently for each occurrence, H or optionally substituted alkyl. In other embodiments, the branch point is glycerol or a glycerol derivative.

[0168] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0169] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0170] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0171] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0172] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0173] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0174] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0175] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0176] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0177] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0178] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0179] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting monomer comprises:

[0180] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting ligand comprises:

[0181] In certain embodiments, the branched linker connecting the carbohydrate-based ligand to the double-stranded iRNA agent can be a branched aliphatic group comprising a group selected from the group consisting of alkyl, amide, disulfide, polyethylene glycol, ether, thioether, hydroxyamino group, and combinations thereof.

[0182] In some embodiments, the bivalent or trivalent branched linker is a lysine-based structure, e.g., [ka] where n is independently 1 to 20, e.g., 1 to 10, 1 to 5, 1 to 3, or 1 to 3. In one embodiment, the trivalent branched linker is [ka] is.

[0183] Exemplary Ligand Monomers In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting monomer comprises:

[0184] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting monomer comprises:

[0185] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting monomer comprises:

[0186] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The targeting monomer comprises:

[0187] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0188] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0189] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0190] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0191] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0192] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0193] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0194] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0195] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0196] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0197] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0198] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0199] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0200] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0201] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0202] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0203] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0204] In some embodiments, both L 2Aand L 2B is different.

[0205] In some preferred embodiments, both L 3A and L 3B is the same.

[0206] In some embodiments, both L 3A and L 3B is different.

[0207] In some preferred embodiments, both L 4A and L 4B is the same.

[0208] In some embodiments, both L 4A and L 4B is different.

[0209] In some preferred embodiments, L 5A , L 5B and L 5C are all the same.

[0210] In some embodiments, L 5A , L 5B and L 5C Two of them are the same.

[0211] In some embodiments, L 5A and L 5B is the same.

[0212] In some embodiments, L 5A and L 5C is the same.

[0213] In some embodiments, L 5B and L 5C is the same.

[0214] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0215] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0216] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0217] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] wherein Y is O or S, and n is 1-6.

[0218] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] wherein Y is O or S, n is 1 to 6, R is hydrogen or a nucleic acid, and R' is a nucleic acid.

[0219] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] wherein Y is O or S, and n is 1-6.

[0220] In certain embodiments, oligomeric compounds described herein, including but not limited to, double-stranded iRNA agents of the invention, have the structure: [ka] wherein Y is O or S, n is 2 to 6, x is 1 to 6, and A is H or a phosphate bond.

[0221] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The compound contains at least one, two, three or four monomers of the formula:

[0222] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] wherein X is O or S.

[0223] In certain embodiments, oligomeric compounds described herein, including but not limited to, double-stranded iRNA agents of the invention, have the structure: [ka] wherein x is 1 to 12.

[0224] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] where R is OH or NHCOCH3.

[0225] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] where R is OH or NHCOCH3.

[0226] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] wherein R is O or S.

[0227] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] where R is OH or NHCOCH3.

[0228] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0229] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] where R is OH or NHCOCH3.

[0230] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] where R is OH or NHCOCH3.

[0231] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] where R is OH or NHCOCH3.

[0232] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] where R is OH or NHCOCH3.

[0233] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The monomers include:

[0234] In the above-described monomers, X and Y are each, independently at each occurrence, H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, a -P(Z')(Z")O-nucleoside, a -P(Z')(Z")O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, a nucleoside, or an oligonucleotide; and Z' and Z" are each, independently at each occurrence, O or S.

[0235] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] is conjugated with a ligand of

[0236] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The ligands include:

[0237] In certain embodiments, a double-stranded iRNA agent has the structure: [ka] The synthesis of the above-mentioned ligands and monomers is described, for example, in U.S. Pat. No. 8,106,022, the entire contents of which are incorporated herein by reference.

[0238] In some embodiments, the double-stranded iRNA agent includes one or more carbohydrate-based ligands conjugated to both ends of the sense strand.

[0239] In some embodiments, the double-stranded iRNA agent includes one or more carbohydrate-based ligands conjugated to both ends of the antisense strand.

[0240] In some embodiments, the double-stranded iRNA agent includes one or more carbohydrate-based ligands conjugated to the 5' or 3' end of the sense strand and one or more carbohydrate-based ligands conjugated to the 5' or 3' end of the antisense strand.

[0241] In some embodiments, the carbohydrate-based ligand is conjugated to the end of the chain via one or more linkers (tethers) and / or carriers.

[0242] In one embodiment, the carbohydrate-based ligands are conjugated to the ends of the chains via one or more linkers (tethers).

[0243] In one embodiment, the carbohydrate-based ligand is conjugated to the 5' end of the sense strand or the antisense strand via a cyclic carrier, optionally via one or more intervening linkers (tethers).

[0244] In some embodiments, a double-stranded iRNA agent includes one or more lipophilic moieties conjugated to one or more internal positions on at least one strand. An internal position on a strand refers to a nucleotide at any position on the strand, excluding the positions from the 3' and 5' ends of the strand (e.g., excluding two positions: position 1 counting from the 3' end and position 1 counting from the 5' end).

[0245] In one embodiment, a double-stranded iRNA agent includes one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, including all but the two most terminal positions on each strand (e.g., four positions: excluding positions 1 and 2 counting from the 3' end and positions 1 and 2 counting from the 5' end). In one embodiment, lipophilic moieties are conjugated to one or more internal positions on at least one strand, including all but the three most terminal positions on each strand (e.g., six positions: excluding positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).

[0246] In one embodiment, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, excluding the cleavage site region of the sense strand, e.g., no lipophilic moieties are conjugated to positions 9-12, counting from the 5' end of the sense strand, e.g., no lipophilic moieties are conjugated to positions 9-11, counting from the 5' end of the sense strand. Alternatively, the internal positions exclude positions 11-13, counting from the 3' end of the sense strand.

[0247] In one embodiment, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, excluding the cleavage site region of the antisense strand, e.g., internal positions excluding positions 12-14 from the 5' end of the antisense strand.

[0248] In one embodiment, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, excluding positions 11-13 on the sense strand, counting from the 3' end, and positions 12-14 on the antisense strand, counting from the 5' end.

[0249] In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5' end of each strand.

[0250] In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0251] In some embodiments, a double-stranded iRNA agent includes one or more lipophilic moieties conjugated to a nucleobase, sugar moiety, or internucleoside linkage of the double-stranded iRNA agent.

[0252] definition Unless otherwise specified, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Some such techniques and procedures are described, for example, in "Carbohydrate Modifications in Antisense Research," Edited by Sangvi and Cook, American Chemical Society, Washington, DC, 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and "Antisense Drug Technology, Principles, Strategies, and Applications," Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A Laboratory Manual," 2002. nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout this disclosure are incorporated herein by reference in their entirety.

[0253] Unless otherwise indicated, the following terms have the following meanings:

[0254] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be regulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNA transcribed from DNA encoding a target protein (including, but not limited to, pre-mRNA and mRNA or portions thereof), and also cDNA and miRNA obtained from such RNA. For example, a target nucleic acid may be a cellular gene (or mRNA transcribed from that gene) whose expression is associated with a particular disorder or pathology. In some embodiments, a target nucleic acid may be a nucleic acid molecule derived from an infectious agent.

[0255] As used herein, the term "iRNA" refers to an agent that mediates targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Agents effective in inducing RNA interference are also referred to herein as siRNAs, RNAi agents, or iRNA agents. Thus, these terms may be used interchangeably herein. As used herein, the term iRNA includes microRNAs and pre-microRNAs. Furthermore, as used herein, a "compound" or "compounds" of the invention also refer to an iRNA agent and may be used interchangeably with an iRNA agent.

[0256] An iRNA agent should include a region sufficiently homologous to a target gene and be of sufficient length, in nucleotides, so that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. (For ease of explanation, the terms nucleotide or ribonucleotide may be used herein in reference to one or more monomeric subunits of an iRNA agent. It will be understood herein that the use of the terms "ribonucleotide" or "nucleotide" herein, in the case of a modified RNA or nucleotide surrogate, can also refer to the modified nucleotide or surrogate replacement moiety at one or more positions.) Thus, an iRNA agent is or includes a region that is at least partially, and in some embodiments, completely, complementary to a target RNA. While perfect complementarity between an iRNA agent and a target is not required, the match must be sufficient to enable the iRNA agent, or its cleavage product, to direct sequence-specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. The degree of complementarity, or homology, with the target strand is most important in the antisense strand. While perfect complementarity is often desirable, particularly in the antisense strand, some embodiments may include one or more, or for example, 6, 5, 4, 3, 2, or fewer, mismatches (with respect to the target RNA), particularly in the antisense strand. The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded nature of the molecule.

[0257] iRNA agents include molecules that are long enough to trigger an interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)); and molecules that are short enough not to trigger an interferon response (which can also be cleaved by Dicer and / or enter RISC), e.g., molecules of a size that allows entry into RISC, e.g., molecules similar to Dicer cleavage products. Molecules that are short enough not to trigger an interferon response are referred to herein as siRNA agents or short iRNA agents. As used herein, "siRNA agent or short iRNA agent" refers to an iRNA agent, e.g., a double-stranded RNA agent or a single-stranded agent, that is short enough not to induce a deleterious interferon response in human cells, e.g., which has a duplex region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or its cleavage product, can downregulate a target gene, for example, by inducing RNAi against the target RNA, where the target can include an endogenous or pathogen target RNA.

[0258] As used herein, a "single-stranded iRNA agent" is an iRNA agent that is composed of a single molecule. It may include a duplex region formed by intrastrand pairing; for example, it may be or include a hairpin or panhandle structure. A single-stranded iRNA agent may be antisense to a target molecule. A single-stranded iRNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of a target mRNA. A single-stranded iRNA agent is at least 14, and in other embodiments, at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.

[0259] A loop refers to a region of an iRNA strand that, when base-pairing with another strand or another portion of the same strand, does not pair with the opposite nucleotide in the duplex.

[0260] Hairpin iRNA agents have a duplex region of at least or equal to 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be 200, 100, or 50 or less in length. In certain embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length. The hairpin has a single-stranded overhang or terminal unpaired region at the 3' end in some embodiments, and in certain embodiments, on the antisense side of the hairpin. In some embodiments, the overhang is 2-3 nucleotides in length.

[0261] As used herein, a "double-stranded (ds) iRNA agent" is an iRNA agent that includes two or more, and in some cases two, strands that can form a region of duplex structure by hybridization between the strands.

[0262] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0263] As used herein, "gene silencing" by an RNA interference molecule refers to a reduction in mRNA levels in a cell of a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between these, of the mRNA levels found in a cell in the absence of the miRNA or RNA interference molecule. In a preferred embodiment, the mRNA levels are reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between 5% and 100%.

[0264] As used herein, the term "modulate gene expression" means up-regulating or down-regulating the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, such that the expression, level, or activity is higher or lower than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," although the use of the word "modulate" is not limited to this definition.

[0265] As used herein, gene expression modulation occurs when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more from that observed in the absence of an siRNA, e.g., an RNAi agent. The percentage and / or fold difference may be calculated relative to a control or non-control, for example, as follows:

number

[0266] As used herein, the terms "inhibit," "down-regulate," or "reduce," with respect to gene expression, mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a modulator. Gene expression is down-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced by at least 10% compared to a corresponding unmodulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably, 100% (i.e., no gene expression).

[0267] As used herein, the terms "increase" or "up-regulate," with respect to gene expression, mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased above that observed in the absence of a modulator. Gene expression is up-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased by at least 10%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more, relative to a corresponding unmodulated control.

[0268] As used herein, the term "increased" or "increasing" generally refers to an increase by a statistically significant amount; for the avoidance of doubt, "increased" means an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference sample, or an increase of up to 100%, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to a reference level, or any increase from 2-fold to 10-fold or more.

[0269] As used herein, the term "reduced" or "reducing" generally refers to a statistically significant reduction. However, for the avoidance of doubt, "reduced" refers to a reduction of at least 10% compared to the reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction of 100% or less (i.e., zero level compared to the reference sample), or any reduction between 10% and 100% compared to the reference level.

[0270] A double-stranded iRNA comprises two oligonucleotide strands sufficiently complementary to hybridize to form a duplex structure. Typically, the duplex structure is 15-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. In some embodiments, longer double-stranded iRNAs, 25-30 base pairs in length, are preferred. In some embodiments, shorter double-stranded iRNAs, 10-15 base pairs in length, are preferred. In another embodiment, the double-stranded iRNA is at least 21 nucleotides in length.

[0271] In some embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity that is complementary to at least a portion of a target sequence, and the duplex region is 14-30 nucleotides in length. Similarly, the region of complementarity to the target sequence is 14-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 nucleotides in length.

[0272] As used herein, the phrase "antisense strand" refers to an oligomeric compound that is substantially or 100% complementary to the intended target sequence. The phrase "antisense strand" includes the antisense region of both oligomeric compounds that are formed from two separate strands, as well as unimolecular oligomeric compounds that can form hairpin or dumbbell structures. The terms "antisense strand" and "guide strand" are used interchangeably.

[0273] The phrase "sense strand" refers to an oligomeric compound having a nucleoside sequence that is wholly or partially identical to a target sequence, such as a sequence of messenger RNA or DNA. The terms "sense strand" and "passenger strand" are used interchangeably.

[0274] "Specifically hybridizable" and "complementary" mean that a nucleic acid can form hydrogen bonds with another nucleic acid sequence, either through Watson-Crick or other non-traditional types. In the context of the nucleic acid molecules of the present invention, the binding free energy between a nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, such as RNAi activity. Determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377; Turner et al., 1987, Am. Chem. Soc. 109: 3783-3785). Percent complementarity refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100% complementarity means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than perfect complementarity refers to a situation in which some (but not all) nucleoside units of two strands can hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected to be non-complementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under the conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions in which the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0275] In some embodiments, the double-stranded region of a double-stranded iRNA agent is at least equal to or equal to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0276] In some embodiments, the antisense strand of a double-stranded iRNA agent is at least equal to or is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0277] In some embodiments, the sense strand of a double-stranded iRNA agent is at least equal to or is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0278] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 15-30 nucleotides in length.

[0279] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 19-25 nucleotides in length.

[0280] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21-23 nucleotides in length.

[0281] In some embodiments, one strand has at least one stretch of 1 to 5 single-stranded nucleotides within the double-stranded region. A "single-stranded nucleotide stretch within a double-stranded region" means that there is at least one nucleotide base pair on both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. When both strands have a stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region, such single-stranded nucleotides can be highly opposite (e.g., a mismatched stretch), or they can be positioned such that the second strand does not have a single-stranded nucleotide opposite the single-stranded iRNA of the first strand, or vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are present within 8 nucleotides from either end, for example, 8, 7, 6, 5, 4, 3, or 2 nucleotides from either the 5' or 3' end of the region of complementarity between the two strands.

[0282] In one embodiment, the double-stranded iRNA agent includes a single-stranded overhang on at least one of its ends, hi one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0283] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide-long single-stranded overhang at the 3' end.

[0284] In some embodiments, each strand of the double-stranded iRNA has a ZXY structure, such as that described in PCT Publication No. WO2004080406, the entire contents of which are incorporated herein by reference.

[0285] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked to each other. The two strands may be linked to each other at both ends or only one end. Linked at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands may be, but are not limited to, (N) n (where N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, where N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker may participate in base-pairing interactions with other nucleotides in the linker. The two strands can also be linked to each other by a non-nucleoside linker, such as the linkers described herein. It will be understood by those skilled in the art that any of the oligonucleotide chemical modifications or alterations described herein can be used in the oligonucleotide linker.

[0286] Hairpin and dumbbell-shaped oligomeric compounds have duplex regions of at least or equal to 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be up to 200, 100, or 50 nucleotide pairs in length. In some embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0287] Hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region, in some embodiments at the 3', and in some embodiments, at the antisense side of the hairpin. In some embodiments, the overhang is 1 to 4, more usually 2 to 3, nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0288] In certain embodiments, two oligomer strands specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0289] As used herein, "stringent hybridization conditions" or "stringent conditions" refers to conditions under which an antisense compound hybridizes to its target sequence but to a minimal number of other sequences. Stringent conditions are sequence-dependent and vary in various circumstances; the "stringent conditions" under which an antisense compound hybridizes to a target sequence are determined by the nature and composition of the antisense compound and the assay in which it is tested.

[0290] It is understood in the art that incorporating nucleotide affinity modifications can allow a greater number of mismatches compared to unmodified compounds.Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, for example, by determining the melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modifications for their ability to increase the melting temperature of RNA:DNA duplexes by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443).

[0291] siRNA design In some embodiments, the double-stranded iRNA agents described in all of the following embodiments, having various siRNA designs, can further include a carbohydrate-based ligand (e.g., GalNAc3). The double-stranded iRNA agents can further include a phosphate mimic as described herein. The double-stranded iRNA agents can further include 2'-OMe modifications to more than 15, more than 20, more than 25, or more than 30 nucleotides.

[0292] In one embodiment, an iRNA agent of the invention is a 19-nt long double-ended bluntmer, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0293] In one embodiment, an iRNA agent of the invention is a 20 nt long blunt-ended duplex, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0294] In one embodiment, an iRNA agent of the invention is a 21 nt long blunt-ended duplex, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0295] In one embodiment, an iRNA agent of the invention comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the iRNA is blunt while the other end comprises a 2-nt overhang. Preferably, the 2-nt overhang is at the 3' end of the antisense strand.

[0296] In one embodiment, the iRNA agent of the invention comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues long and, starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues long and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; wherein the 5'-end of the antisense strand is 10 to 30 ribonucleotides that are not paired with the sense strand. The double-stranded nucleic acid comprises at least one motif of three 2'-F modifications in three consecutive nucleotides, thereby forming a 10-30 nucleotide single-stranded 5' overhang; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so as to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides, wherein at least one of the motifs is located at or near the cleavage site; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

[0297] In one embodiment, an iRNA agent of the invention includes a sense strand and an antisense strand, wherein the iRNA agent includes a first strand having a length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand including 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 the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1-4 nucleotides longer than the first strand at its 3' end, the duplex region is at least 25 nucleotides long, and the second strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the second strand such that the iRNA agent reduces target gene expression when introduced into a mammalian cell, and wherein Dicer cleavage of the iRNA preferentially results in an siRNA including the 3' end of the second strand, thereby reducing target gene expression in a mammal.

[0298] In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs is at the cleavage site of the sense strand. For example, the sense strand can contain at least one motif of three 2'-F modifications in three consecutive nucleotides within positions 7-15 from the 5' end.

[0299] In one embodiment, the antisense strand of an iRNA agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs is at or near the cleavage site of the antisense strand. For example, the antisense strand can contain at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides within positions 9-15 from the 5' end.

[0300] For iRNA agents having a duplex region 17-23 nt in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be located 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 of the antisense strand, or from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the iRNA from the 5' end.

[0301] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand includes at least two motifs of three identical modifications in three consecutive nucleotides, where at least one of the motifs is at or near the cleavage site within the strand, and at least one of the motifs is in another portion of the strand separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand can also include at least one motif of three identical modifications in three consecutive nucleotides, where at least one of the motifs is at or near the cleavage site within the strand. The modifications in the motifs at or near the cleavage site in the sense strand are different from the modifications in the motifs at or near the cleavage site in the antisense strand.

[0302] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, where the sense strand includes at least one motif of three 2'-F modifications in three consecutive nucleotides, where at least one of the motifs is at or near the site of cleavage in the strand. In one embodiment, the antisense strand also includes at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the site of cleavage.

[0303] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, where the sense strand includes at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand includes at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0304] In one embodiment, the iRNA agent of the present invention contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., relative to the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

[0305] In one embodiment, an iRNA agent of the invention includes 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, which may be independently selected from the group A:U, G:U, I:C, and a mismatch pair, e.g., a non-canonical or non-canonical pairing or a pairing including a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0306] In one embodiment, the nucleotide at position 1 from the 5' end of the antisense strand into the duplex region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs from the 5' end of the antisense strand into the duplex region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand into the duplex region is an AU base pair.

[0307] In some embodiments, a double-stranded RNA (dsRNA) agent comprises a sense strand and an antisense strand, each strand having between 14 and 40 nucleotides. The dsRNA agent is represented by formula (I): [ka]

[0308] In Formula (I), 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 one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification or a 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0309] C1 is a thermolabile nucleotide located at the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is located in the sense strand at a position that pairs with nucleotides 2-8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 of the 5' end of the sense strand. The C1 nucleotide has a thermolabile modification that can include an abasic modification; a mismatch with the opposing nucleotide of the duplex; and a sugar modification, such as a 2'-deoxy modification, or an acyclic nucleotide, such as an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In one embodiment, C1 has (i) a mismatch with the opposing nucleotide of the antisense strand; (ii) an abasic modification selected from the group consisting of: [ka] and (iii) a sugar modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, 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; optionally, at least one nucleobase in the mismatch pair is a 2'-deoxy-nucleobase. In one example, the thermolabilizing modification in C1 is GNA or [ka] is.

[0310] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides the nucleotide with steric bulk equal to or less than that of a 2'-OMe modification. Steric bulk refers to the total steric effect of the modification. Methods for determining the steric effect of a nucleotide modification are known to those skilled in the art. The modification may be a modification at the 2' position of the ribose sugar of the nucleotide, or a modification of the backbone of a non-ribose nucleotide, an acyclic nucleotide, or a similar or equivalent modification of the 2' position of the ribose sugar, and provides the nucleotide with steric bulk equal to or less than that of a 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 one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA.

[0311] n 1 , n 3 , and q 1are independently 4 to 15 nucleotides in length.

[0312] n 5 , q 3 , and q 7 are independently 1 to 6 nucleotides in length.

[0313] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length; or 4 is 0.

[0314] q 5 are independently 0 to 10 nucleotides in length.

[0315] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0316] Or, n 4 is 0 to 3 nucleotides in length.

[0317] In one embodiment, n 4 can be 0. In one 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, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0318] In one embodiment, n 4 , q 2 , and q 6 are each 1.

[0319] In one embodiment, n2 , n 4 , q 2 , q 4 , and q 6 are each 1.

[0320] In one embodiment, when the sense strand is 19 to 22 nucleotides in length, C1 is at positions 14 to 17 of the 5' end of the sense strand, and n 4 is 1. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0321] In one embodiment, T3' begins at position 2 of the 5' end of the antisense strand. In one example, T3' is at position 2 of the 5' end of the antisense strand, and 6 is equal to 1.

[0322] In one embodiment, T1' begins at position 14 of the 5' end of the antisense strand. In one example, T1' is at position 14 of the 5' end of the antisense strand and 2 is equal to 1.

[0323] In an exemplary embodiment, T3' begins at position 2 of the 5' end of the antisense strand, and T1' begins at position 14 of the 5' end of the antisense strand. In one example, T3' begins at position 2 of the 5' end of the antisense strand, and q 6 is equal to 1, T1' starts from position 14 of the 5' end of the antisense strand, and q 2 is equal to 1.

[0324] In one embodiment, T1' and T3' are separated by 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0325] In one embodiment, T1' is at position 14 of the 5' end of the antisense strand. 2 is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose.

[0326] In one embodiment, T3' is at position 2 of the 5' end of the antisense strand. In one example, T3' is at position 2 of the 5' end of the antisense strand, and 6 is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides steric bulk less than or equal to that of 2'-OMe ribose.

[0327] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, when the sense strand is 19-22 nucleotides long, T1 is at position 11 of the 5' end of the sense strand, n 2 is 1. In an exemplary embodiment, when the sense strand is 19-22 nucleotides in length, T1 is at the cleavage site of the sense strand at position 11 at the 5' end of the sense strand, and n 2 is 1.

[0328] In one embodiment, T2' begins at position 6 of the 5' end of the antisense strand. In one example, T2' is from position 6 to position 10 of the 5' end of the antisense strand, and 4 is 1.

[0329] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., at position 11 of the 5' end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n 2 is 1; T1' is at position 14 of the 5' end of the antisense strand, and q 2 is equal to 1, and the 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 located at positions 6-10 of the 5' end of the antisense strand, and q 4 is 1; and T3' is at position 2 of 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 less than or equal to that of 2'-OMe ribose.

[0330] In one embodiment, T2' begins at position 8 of the 5' end of the antisense strand. 4 is 2.

[0331] In one embodiment, T2' begins at position 9 of the 5' end of the antisense strand. 4 is 1.

[0332] In one embodiment, 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; 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0333] In one embodiment, 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 q7 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0334] In one embodiment, 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.

[0335] In one embodiment, 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'-OMe, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0336] In one embodiment, 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.

[0337] In one embodiment, 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 q3 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0338] In one embodiment, 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.

[0339] In one embodiment, 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, 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; 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0340] In one embodiment, 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.

[0341] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 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 has at least two additional TTs at the 3' end of the antisense strand; 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), as well as two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0342] In one embodiment, 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.

[0343] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0344] In one embodiment, 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 6is 1, B4' is 2'-F, and q 7 is 1.

[0345] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0346] In one embodiment, 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.

[0347] In one embodiment, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0348] The dsRNA agent can include a phosphorus-containing group, such as a phosphate or phosphate mimic, at the 5'-end of either the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal 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 a 5'-E-VP isomer (i.e., trans-vinyl phosphate, [ka] ), or the 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ) or a mixture thereof.

[0349] In one embodiment, the dsRNA agent includes a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the dsRNA agent includes a phosphorus-containing group at the 5'-end of the antisense strand.

[0350] In one embodiment, the dsRNA agent includes a 5'-P. In one embodiment, the dsRNA agent includes a 5'-P in the antisense strand.

[0351] In one embodiment, the dsRNA agent comprises a 5'-PS.In one embodiment, the dsRNA agent comprises a 5'-PS on the antisense strand.

[0352] In one embodiment, the dsRNA agent comprises a 5'-VP. In one embodiment, the dsRNA agent comprises a 5'-VP in the antisense strand. In one embodiment, the dsRNA agent comprises a 5'-E-VP in the antisense strand. In one embodiment, the dsRNA agent comprises a 5'-Z-VP in the antisense strand.

[0353] In one embodiment, the dsRNA agent comprises a 5'-PS2.In one embodiment, the dsRNA agent comprises a 5'-PS2 in the antisense strand.

[0354] In one embodiment, the dsRNA agent comprises a 5'-PS2. In one embodiment, the dsRNA agent comprises a 5'deoxy5'-C-malonyl in the antisense strand.

[0355] In some embodiments, the phosphate mimetic has formula PM-I: [ka] is represented by During the ceremony: R c and R d are each independently selected from CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group; B is a natural nucleobase, a modified nucleobase, a universal base, or is absent; R 10 is a phosphoramidite; X2 is OH, F, OCH3, or OCH2CH2OCH3, and R8 is absent; or X2 is O, R8 is a glutathione-sensitive moiety.

[0356] In certain embodiments, B is a natural nucleobase.

[0357] In certain embodiments, R c and R d are each independently selected from CH3;

[0358] In certain embodiments, X2 is F or OCH3 and R8 is absent.

[0359] In certain embodiments, X2 is O and R8 is a glutathione-sensitive moiety.

[0360] In certain embodiments, R c and R d is CH3, R8 is absent, and X2 is F or OCH3.

[0361] In certain embodiments, R c and R d is CH2CH3, R8 is absent, and X2 is F or OCH3.

[0362] In certain embodiments, the phosphate mimetic has formula PM-II: [ka] is represented by During the ceremony: B is a natural nucleobase, a modified nucleobase, a universal base, or is absent; R 10 is a phosphoramidite; X2 is OH, F, OCH3, or OCH2CH2OCH3.

[0363] In certain embodiments, B is a natural nucleobase.

[0364] In certain embodiments, X2 is F or OCH3.

[0365] In certain embodiments, the phosphate mimetic has formula PM-III: [ka] is represented by During the ceremony: B is a natural nucleobase, a modified nucleobase, a universal base, or is absent; R 10 is a phosphoramidite; X2 is OH, F, OCH3, or OCH2CH2OCH3.

[0366] In certain embodiments, B is a natural nucleobase.

[0367] In certain embodiments, X2 is F or OCH3.

[0368] In some embodiments, the phosphate mimetic has formula PM-IV: [ka] is represented by During the ceremony: R c and R dare each independently selected from CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group; V is O; Z1 is a nucleoside comprising a phosphoramidite and a sugar moiety; V is attached to the 4'-carbon of the sugar moiety.

[0369] Typically, the sugar moiety is a furanose and V is attached to the 4'-carbon of the furanose.

[0370] In certain embodiments, R c and R d is CH3. In certain embodiments, R c and R d is CH2CH3.

[0371] Further exemplary phosphate mimetics suitable for the double-stranded iRNA agents herein can be found in WO 2018 / 045317; U.S. Pat. No. 8,927,513; and U.S. Pat. No. 11,119,136, the entire contents of which are incorporated herein by reference.

[0372] In one embodiment, 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. The dsRNA agent also includes a 5'-PS.

[0373] In one embodiment, 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 dsRNA agent also includes a 5'-P.

[0374] In one embodiment, 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 dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0375] In one embodiment, 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 dsRNA agent also includes a 5'-PS2.

[0376] In one embodiment, 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 dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0377] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0378] In one embodiment, 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 6is 1, B4' is 2'-OMe, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0379] In one embodiment, 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, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0380] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS2.

[0381] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0382] In one embodiment, 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 dsRNA agent also includes a 5'-P.

[0383] In one embodiment, 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'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0384] In one embodiment, 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 dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0385] In one embodiment, 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 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0386] In one embodiment, 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 dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0387] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0388] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0389] In one embodiment, 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; 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0390] In one embodiment, 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 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. The dsRNA agent also includes a 5'-PS2.

[0391] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0392] In one embodiment, 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'-F, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0393] In one embodiment, 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 dsRNA agent also includes a 5'-PS.

[0394] In one embodiment, 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'-F, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0395] In one embodiment, 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 dsRNA agent also includes a 5'-PS2.

[0396] In one embodiment, 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, 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 dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0397] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0398] In one embodiment, 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, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0399] In one embodiment, 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 1-5 of the sense strand (counting from the 5' end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0400] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS2.

[0401] In one embodiment, 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; 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0402] In one embodiment, 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 dsRNA agent also includes a 5'-P.

[0403] In one embodiment, 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 dsRNA agent also includes a 5'-PS.

[0404] In one embodiment, 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 dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0405] In one embodiment, 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 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 dsRNA agent also includes a 5'-PS2.

[0406] In one embodiment, 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 dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0407] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0408] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0409] In one embodiment, 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, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0410] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS2.

[0411] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0412] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of a dsRNA agent of the invention is modified. For example, if a dsRNA agent is 50% modified, then 50% of all nucleotides present in the dsRNA agent contain a modification as described herein.

[0413] In one embodiment, the sense and antisense strands of a dsRNA agent are each independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0414] In one embodiment, the sense and antisense strands of the dsRNA agent each contain at least two different modifications.

[0415] In one embodiment, the dsRNA agent of Formula (I) further comprises a 3' and / or 5' overhang of 1 to 10 nucleotides in length. In one example, the dsRNA agent of Formula (I) comprises a 3' overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In another example, the dsRNA agent has a 5' overhang at the 5' end of the sense strand.

[0416] In one embodiment, a dsRNA agent of the invention does not include any 2'-F modifications.

[0417] In one embodiment, the sense strand and / or antisense strand of a dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0418] In one embodiment, the sense and antisense strands of the dsRNA agent each have 15 to 30 nucleotides. In one example, the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0419] In one embodiment, the nucleotide at position 1 of the 5' end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pairs from the 5' end of the antisense strand is an AU base pair.

[0420] In one embodiment, the antisense strand of the dsRNA agent of the present invention is 100% complementary to the target RNA for hybridizing to the target RNA and inhibiting its expression by RNA interference. In another embodiment, the antisense strand of the dsRNA agent of the present invention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0421] In one aspect, the present invention relates to a dsRNA agent, as defined herein, capable of inhibiting expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand comprises at least one thermolabile nucleotide, at least one of which is located opposite or near the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). Each of the embodiments and aspects described herein relating to a dsRNA represented by Formula (I) can also be applied to a dsRNA comprising a thermolabile nucleotide.

[0422] For example, if the sense strand is 21 nucleotides long, the thermally destabilizing nucleotide may be located between positions 14 and 17 at the 5' end of the sense strand. The antisense strand includes at least two modified nucleic acids that are less than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids that are less than sterically demanding 2'-OMe are spaced 11 nucleotides apart. For example, the two modified nucleic acids are located at positions 2 and 14 at the 5' end of the antisense strand.

[0423] In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand may be a bivalent or trivalent branched linker, such as: [ka] In one example, the ASGPR ligand is added to the 3' or 5' end of the sense strand.

[0424] For example, a dsRNA agent as defined herein can include: i) a phosphorus-containing group at the 5'-end of the sense strand or antisense strand; ii) two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5'-end of the antisense strand) of the antisense strand; and iii) a ligand, e.g., an ASGPR ligand (e.g., one or more GalNAc derivatives attached directly or via a bivalent or trivalent branched linker), at the 5'-end or 3'-end of the sense strand or antisense strand. For example, the ligand can be at the 3'-end or 5'-end of the sense strand.

[0425] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA 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' or 5' end of the sense strand.

[0426] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA 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' or 5' end of the sense strand.

[0427] In one embodiment, 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 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, 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 dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' or 5' end of the sense strand.

[0428] In one embodiment, 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, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA 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' or 5' end of the sense strand.

[0429] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 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' or 5' end of the sense strand.

[0430] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA 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' or 5' end of the sense strand.

[0431] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA 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' or 5' end of the sense strand.

[0432] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' or 5' end of the sense strand.

[0433] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA 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' or 5' end of the sense strand.

[0434] In one embodiment, 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, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end) of the antisense strand. The dsRNA agent also includes a 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' or 5' end of the sense strand.

[0435] In one embodiment, 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 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, 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 dsRNA 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' or 5' end of the sense strand.

[0436] In one embodiment, 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; 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA 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' or 5' end of the sense strand.

[0437] In one embodiment, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' or 5' end of the sense strand.

[0438] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA 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' or 5' end of the sense strand.

[0439] In one embodiment, 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, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 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' or 5' end of the sense strand.

[0440] In one embodiment, 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; 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, 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 dsRNA 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' or 5' end of the sense strand.

[0441] In one embodiment, 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 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA 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' or 5' end of the sense strand.

[0442] In one embodiment, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' or 5' end of the sense strand.

[0443] In one embodiment, 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, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA 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' or 5' end of the sense strand.

[0444] In one embodiment, 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, and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The dsRNA agent also includes a 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' or 5' end of the sense strand.

[0445] In one particular embodiment, a dsRNA agent of the invention: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by a bivalent or trivalent branched linker; and (iii) a sense strand having 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21 (counting from the 5' end) and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14-16, 18, and 20; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23 (counting from the 5' end), and 2'F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 (counting from the 5' end) and between nucleotide positions 22 and 23; The dsRNA agent has an antisense strand with a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0446] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21 (counting from the 5' end) and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent comprises an antisense strand with a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0447] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21 (counting from the 5' end), 2'-F modifications at positions 7 and 9, and a deoxy-nucleotide (e.g., dT) at position 11; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA 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.

[0448] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA 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.

[0449] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (iii) 2'-OMe modifications at positions 1 to 9 and 12 to 21, and 2'-F modifications at positions 10 and 11; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 (counting from the 5' end) and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA 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.

[0450] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA 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.

[0451] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 25 nucleotides long; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23 (counting from the 5' end), 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxy-nucleotides (e.g., dT) at positions 24 and 25; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; This dsRNA agent has a four nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0452] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA 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.

[0453] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA 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.

[0454] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 19 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2'-F modifications at positions 5 and 7 to 9; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 21 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21; The dsRNA 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.

[0455] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand having: (i) 18–23 nucleotides long; (ii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (iii) three consecutive 2'-F modifications at positions 7 to 15; and (b) an antisense strand having: (i) 18–23 nucleotides long; (ii) at least a 2'-F modification anywhere in the strand; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); Includes; wherein the dsRNA agent optionally has one or more lipophilic moieties conjugated to one or more positions in at least one strand; Here, the dsRNA agent has a blunt end at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or two nucleotide overhangs at the blunt ends at both ends of the duplex.

[0456] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand having: (i) 18–23 nucleotides long; (ii) fewer than four 2′-F modifications; (iii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (b) an antisense strand having: (i) 18–23 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); Includes; wherein the dsRNA agent optionally has one or more lipophilic moieties conjugated to one or more positions in at least one strand; Here, the dsRNA agent has a blunt end at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or two nucleotide overhangs at the blunt ends at both ends of the duplex.

[0457] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand having: (i) 19–35 nucleotides long; (ii) fewer than four 2′-F modifications; (iii) ASGPR ligands attached to the 3' or 5' end, comprising one, two, or three GalNAc derivatives attached by bivalent or trivalent branched linkers; (b) an antisense strand having: (i) 19–35 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); Includes; wherein the duplex region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); and wherein the dsRNA agent optionally has one or more lipophilic moieties conjugated to one or more positions in at least one strand; Here, the dsRNA agent has a blunt end at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or two nucleotide overhangs at the blunt ends at both ends of the duplex.

[0458] In one embodiment, a dsRNA agent of the invention has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has an ASGPR ligand attached on at least one strand, the ASGPR ligand comprising one, two, or three GalNAc derivatives attached via a bivalent or trivalent branched linker; and wherein the dsRNA agent has less than 20%, less than 15%, and less than 10% non-natural nucleotides.

[0459] Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F, and others.

[0460] In one embodiment, a dsRNA agent of the invention has sense and antisense strands having lengths of 15-30 nucleotides; comprises at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplexed region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has an ASGPR ligand attached on at least one strand comprising three GalNAc derivatives attached via a trivalent branched linker; and wherein the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% natural nucleotides, e.g., 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides.

[0461] In one embodiment, a dsRNA agent of the invention has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); wherein the dsRNA agent has an ASGPR ligand attached on at least one strand comprising three GalNAc derivatives attached via a trivalent branched linker; and wherein the dsRNA agent has 100% natural nucleotides, e.g., 2'-OH, 2'-deoxy, and 2'-OMe are natural nucleotides.

[0462] In one embodiment, a dsRNA agent of the invention includes a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand sequence is represented by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) During the ceremony: i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b represents an oligonucleotide sequence containing, independently, 1, 2, 3, 4, 5, or 6 modified nucleotides; each n p and n q independently represent overhanging nucleotides; N b and Y do not have the same modification; XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides; The dsRNA agent has attached on at least one strand an ASGPR ligand comprising one, two, or three GalNAc derivatives attached via a bivalent or trivalent branched linker; The antisense strand of the dsRNA contains two blocks of 1, 2, or 3 phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0463] Various publications describe multimeric siRNAs, all of which may be used with the iRNAs of the present invention, including WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire contents of which are incorporated herein by reference.

[0464] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the iRNA agents of the invention are modified.

[0465] In some embodiments, each of the sense and antisense strands of an iRNA agent is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0466] In some embodiments, the sense and antisense strands of an iRNA agent each contain at least two different modifications.

[0467] In some embodiments, the double-stranded iRNA agents of the invention do not include any 2'-F.

[0468] In some embodiments, a double-stranded iRNA agent of the invention includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. In one example, a double-stranded iRNA agent of the invention includes 9 or 10 2'-F modifications.

[0469] The iRNA agent of the present invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may be present at any nucleotide in the sense strand, antisense strand, or both strands at any position. For example, the internucleotide bond modification may be present at every nucleotide in the sense strand or antisense strand; each internucleotide bond modification may be present in an alternating pattern in the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification in the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of the internucleotide bond modification in the sense strand may have a shift relative to the alternating pattern of the internucleotide bond modification in the antisense strand.

[0470] In one embodiment, the iRNA agent includes a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can contain two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between them. The internucleotide bond modification can also be formed to link the overhang nucleotide to the terminal base-pairing nucleotide in the duplex region. For example, at least two, three, four, or all of the overhanging nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, an additional phosphorothioate or methylphosphonate internucleotide bond can be present linking the overhang nucleotide to the next base-pairing nucleotide. For example, at least two phosphothioate internucleotide bonds can be present between the terminal three nucleotides, two of which are overhanging nucleotides and the third nucleotide is the next base-pairing nucleotide to the overhanging nucleotide. Preferably, these terminal three nucleotides can be at the 3' end of the antisense strand.

[0471] In some embodiments, the sense strand and / or antisense strand of an iRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0472] In some embodiments, the antisense strand of an iRNA agent of the invention is 100% complementary to the target RNA in order to hybridize to the target RNA and inhibit its expression by RNA interference, hi other embodiments, the antisense strand of an iRNA agent of the invention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

[0473] In one aspect, the present invention relates to a double-stranded iRNA agent capable of inhibiting expression of a target gene. The iRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand includes at least one thermolabile nucleotide, where the at least one thermolabile nucleotide is located at or near the opposite site from the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). For example, if the sense strand is 21 nucleotides long, the thermolabile nucleotide is located at positions 14 to 17 of the 5' end of the sense strand. The antisense strand includes at least two modified nucleic acids that are smaller than sterically bulky 2'-OMe modifications. Preferably, the two modified nucleic acids that are smaller than sterically bulky 2'-OMe modifications are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0474] In some embodiments, the compounds of the present invention disclosed herein are miRNA mimics. In one design, miRNA mimics are double-stranded molecules (e.g., having a duplex region of about 16 to about 31 nucleotides in length) and contain one or more sequences that share identity with the mature strand of a given miRNA. Double-stranded miRNA mimics have designs similar to those described above for double-stranded iRNAs. In some embodiments, miRNA mimics contain a 16-31 nucleotide duplex region and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide) and all Cs and Us; the antisense strand modifications may include 2'F modifications of all Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and a stable internucleotide linkage coupled with a two-nucleotide 3' overhang.

[0475] In some embodiments, the compounds of the present invention disclosed herein are antimirs. In some embodiments, the compounds of the present invention comprise at least two antimers covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as those described in this disclosure. The terms "antimer," "microRNA inhibitor," or "miR inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the activity of a specific miRNA. Inhibitors can take various forms, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplexes), and hairpin designs. Generally, microRNA inhibitors comprise one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand(s) of the targeted miRNA. Additionally, miRNA inhibitors may also comprise additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA. The additional sequence can be the reverse complement of the sequence flanking the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequence can be any sequence (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences is any sequence capable of forming a hairpin. Thus, in some embodiments, a sequence that is the reverse complement of an miRNA is flanked on the 5' and 3' sides by hairpin structures. When the microRNA inhibitor is double-stranded, it can contain mismatches between nucleotides in the opposite strands. Additionally, the microRNA inhibitor can be linked to a conjugate moiety to facilitate uptake of the inhibitor into a cell.

[0476] MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13:723-730 (2007) and WO 2007 / 095387 and WO 2008 / 036825, the entire contents of each of which are incorporated herein by reference. One skilled in the art can select a sequence from a database for a desired miRNA and design an inhibitor useful in the methods disclosed herein.

[0477] In some embodiments, the compounds of the present invention disclosed herein are antagomirs. In some embodiments, the compounds of the present invention comprise at least two antagomirs, covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, e.g., a linker described herein. Antagomirs are RNA-like oligonucleotides with various modifications for RNAse protection and pharmacological properties, such as improved tissue and cellular uptake. They differ from conventional RNAs by, for example, complete 2'-O-methylation of sugars, phosphorothioate intersugar linkages, and, for example, a cholesterol moiety at the 3' end. In a preferred embodiment, the antagomir contains 2'-O-methyl modifications at all nucleotides, a cholesterol moiety at the 3' end, two phosphorothioate intersugar linkages at the first two positions at the 5' end, and four phosphorothioate linkages at the 3' end of the molecule. Antagomirs can be used to efficiently silence endogenous miRNAs by forming a duplex containing the antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122, as described in Krutzfeldt et al., Nature, 2005, 438:685-689, the entire contents of which are expressly incorporated herein by reference.

[0478] Recent studies have found that dsRNA can also activate gene expression, a mechanism known as "small RNA-induced gene activation" or RNAa (activating RNA). See, for example, Li, LC et al. Proc Natl Acad Sci USA (2006), 103(46):17337-42 and Li LC (2008). "Small RNA-Mediated Gene Activation". RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press. ISBN 978-1-904455-25-7. It has been shown that dsRNA-targeted gene promoters induce strong transcriptional activation of associated genes. Endogenous miRNAs that cause RNAa are also found in humans. Check E. Nature (2007). 448(7156):855-858.

[0479] Another surprising observation is that gene activation by RNAa is long-lasting. Induction of gene expression has been observed to continue for more than 10 days. The persistent effect of RNAa may be due to epigenetic changes at the dsRNA target site. In some embodiments, RNA activators can increase gene expression. In some embodiments, increased gene expression inhibits survival, growth, development, and / or reproduction.

[0480] Thus, in some embodiments, the compounds of the invention disclosed herein are activator-RNAs. In some embodiments, the compounds of the invention comprise at least two activator-RNAs covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, e.g., a linker described in this disclosure.

[0481] Therefore, in some embodiments, the compounds of the present invention disclosed herein are triplex-forming oligonucleotides (TFOs). In some embodiments, the compounds of the present invention comprise at least two TFOs that are covalently linked to each other or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, for example, a linker described in the present disclosure. Recent studies have shown that triplex-forming oligonucleotides can be designed that can recognize and bind to polypurine / polypyrimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, LJ, et al., Science (1989) vol. 245, pp. 725-730; Moser, HE, et al., Science (1987) vol. 238, pp. 645-630; Beal, PA, et al., Science (1992) vol. 251, pp. 1360-1363; Conney, M., et al., Science (1988) vol. 241, pp. 456-459, and Hogan, ME et al., EP Publication No. 375408. Modifications of oligonucleotides, such as the introduction of intercalating agents and intersugar bond replacement, as well as optimization of binding conditions (pH and cation concentration), have helped overcome the inherent obstacles to TFO activity, such as charge repulsion and instability, and it has recently been shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review, see Seidman and Glazer, J Clin Invest 2003;1 12:487-94). Generally, triplex-forming oligonucleotides have the sequence match: Oligo 3'-AGGT Double-stranded 5'-AGCT Double-stranded 3'-TCGA

[0482] However, A-AT and G-GC triplets have been shown to have the highest triple helix stability (Reither and Jeltsch, BMC Biochem, 2002, Sept. 12, Epub). The same authors demonstrated that TFOs designed according to the A-AT and G-GC rules did not form nonspecific triplexes, indicating that triplex formation is indeed sequence-specific.

[0483] Thus, for any given sequence, triplex-forming sequences can be devised. Triplex-forming oligonucleotides are preferably at least 15, more preferably 25, and even more preferably 30 or more nucleotides in length, up to 50 or 100 nucleotides.

[0484] The formation of triple helix structures with target DNA induces conformational and functional changes, blocking transcription initiation and elongation, allowing for the introduction of desired sequence changes in endogenous DNA, resulting in specific downregulation of gene expression. Examples of such suppression of gene expression in TFO-treated cells include knockout of the episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri, et al., J Biol Chem, 2001;276:28991-98), as well as sequence-specific and target-specific downregulation of the expression of the Ets2 transcription factor, which is important in prostate cancer pathogenesis (Carbone, et al., Nucl Acids Res. 2003;31:833-43), and the inflammatory ICAM-I gene (Besch et al., J Biol Chem, 2002;277:32473-79). Furthermore, Vuyisich and Beal recently showed that sequence-specific TFOs can bind to dsRNA and inhibit the activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

[0485] Furthermore, TFOs designed according to the above principles can induce directed mutagenesis capable of DNA repair, thereby providing both down- and up-regulation of endogenous gene expression (Seidman and Glazer, J Clin Invest 2003;112:487-94).Detailed descriptions of the design, synthesis, and administration of effective TFOs can be found in U.S. Patent Application Publication Nos. 2003-017068 and 2003-0096980 to Froehler et al., 2002-0128218 and 2002-0123476 to Emanuele et al., and U.S. Patent No. 5,721,138 to Lawn, the entire contents of which are incorporated herein by reference.

[0486] Nucleic acid modification In some embodiments, the double-stranded iRNA agent of the present invention comprises at least one nucleic acid modification described herein. For example, the at least one modification is selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof. Without limitation, such a modification can be present anywhere in the double-stranded iRNA agent of the present invention. For example, the modification can be present in one of the RNA molecules.

[0487] Nucleic acid modifications (nucleobases) The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common types of such heterocyclic bases are purines and pyrimidines. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are generally referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3'-5' phosphodiester bond.

[0488] In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art can be used in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) and any one of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used. When a natural base is substituted with an unnatural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases, including conjugate moieties, e.g., ligands, as described herein. Preferred conjugate moieties for conjugation to nucleobases include cationic amino groups, which can be conjugated to the nucleobases via linkers having suitable alkyl, alkenyl, or amide bonds.

[0489] The oligomeric compounds described herein may also include 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). Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6-(Isopentenyl)adenine, 6-(Alkyl)adenine, 6-(Methyl)adenine, 7-(Deaza)adenine, 8-(Alkenyl)adenine, 8-(Alkyl)adenine, 8-(Alkynyl)adenine, 8-(Amino)adenine, 8-(Halo)adenine, 8-(Hydroxyl)adenine, 8-(Thioalkyl)adenine, 8-(Thiol)adenine, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 , N 6 -(Dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine cytosine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(Acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil , 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil Uracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil Uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl allyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stivenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N, 2 -substituted purines, N6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used.

[0490] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an iRNA duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, and imidizopyridinyl. In some embodiments, the aryl groups include phenyl, 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-propynylisocarbostyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stibenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0491] No. 3,687,808; those disclosed in International Application No. PCT / US09 / 038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30,613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P. Ed. Wiley-VCH, 2008; and those disclosed by Sanghvi, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, all of which are incorporated herein by reference.

[0492] In certain embodiments, the modified nucleobase is a nucleobase that is structurally very similar to the parent nucleobase, such as, for example, 7-deazapurine, 5-methylcytosine, or G-clamp. In certain embodiments, the nucleobase mimic comprises a more complex structure, such as, for example, a tricyclic phenoxazine nucleobase mimic. Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.

[0493] Nucleic acid modification (sugar) The double-stranded iRNA agents of the invention provided herein can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers, including nucleosides or nucleotides, having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, including, but not limited to, the addition of a substituent, or the bridging of two non-geminal ring atoms to form a locked nucleic acid or a bicyclic nucleic acid. In certain embodiments, an oligomeric compound includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers that are LNAs.

[0494] In some embodiments of the locked nucleic acid, the 2' position of the furanosyl is -[C(R1)(R2)] n -, -[C(R1)(R2)] n -O-, -[C(R1)(R2)] n -N(R1)-, -[C(R1)(R2)] n -N(R1)-O-, -[C(R1R2)] n -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -C(=NR1)-, -C(=NR1)- O-, -C(=O)-, -C(=O)O-, -C(=S)-, -C(=S)O-, -C(=S)S-, -O-, -Si(R1)2-, -S(=O) x - and -N(R1)-; During the ceremony: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R1 and R2 is independently H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocyclic radical, a substituted heterocyclic radical, heteroaryl, substituted heteroaryl, a C5-C7 cycloaliphatic radical, a substituted C5-C7 cycloaliphatic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); Each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0495] In some embodiments, each of the linkers in the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -C(R1R2)-N(R1)-O-, or -C(R1R2)-ON(R1)-. In other embodiments, each of the linkers is independently 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or a C1-C12 alkyl.

[0496] Several LNAs have been prepared and disclosed in the patent and scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh et al. al., J. Org. Chem., 1998, 63, 10035-10039; examples of issued U.S. patents and published applications disclosing LNAs include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Patent Application Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0497] Also provided herein are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-O-2') bond to form a bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage may be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, and the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; if there is an ethylene group at this position, the term ethyleneoxy(4'-CH2CH2-O-2')LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456; Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy(4'-CH2-O-2')LNA and other bicyclic sugar analogs exhibit very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility properties. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0498] A similarly described isomer of methyleneoxy(4'-CH2-O-2')LNA is α-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have superior stability against 3'-exonucleases. α-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras, which have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0499] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0500] Analogs of methyleneoxy(4'-CH2-O-2')LNA, phosphorothioate-methyleneoxy(4'-CH2-O-2')LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of a novel conformationally restricted, high-affinity oligonucleotide analog, 2'-amino-LNA, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been reported.

[0501] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes, but is not limited to, bicyclic modified sugars, including methyleneoxy (4'-CH2-O-2') LNA and ethyleneoxy (4'-(CH2)2-O-2'-bridged) ENA; substituted sugars, particularly 2'-substituted sugars having a 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituent; and 4'-thio modified sugars. Sugars can also be substituted with sugar mimetic groups, among others. Methods for preparing modified sugars are well known to those of skill in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos. 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; and 5,576,427. Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO 2005 / 121371.

[0502] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) nCH2CH2OR, n=1-50; "locked" nucleic acids (LNA) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2) n AMINE (n=1-10, AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0503] "Deoxy" modifications include hydrogen (i.e., deoxyribose sugars particularly associated with single-stranded overhangs); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n Included are CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl, and alkynyl, which may be optionally substituted, for example, with an amino function.

[0504] Other suitable 2'-modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0505] Modifications at the 2' position can be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent on C2' of the ribose in the same configuration as the 2'-OH in arabinose.

[0506] A sugar may contain two different modifications, e.g., gem modifications, at the same carbon of the sugar. The sugar group may also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, an oligomeric compound may contain, for example, one or more monomers containing arabinose as the sugar. The monomer may have an α-linkage at the 1'-position of the sugar, e.g., an α-nucleoside. The monomer may also have the opposite configuration at the 4'-position, e.g., C5' and H4' or the substituents replacing them are interchanged. When C5' and H4' or the substituents replacing them are interchanged, the sugar is said to be modified at the 4'-position.

[0507] The double-stranded iRNA agents of the invention disclosed herein can also contain abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having another chemical group at C1' in place of a nucleobase. See, e.g., U.S. Pat. No. 5,998,203, the entire contents of which are incorporated herein. These abasic sugars can also contain further modifications to one or more of the constituent sugar atoms. The double-stranded iRNA agents of the invention can also contain one or more sugars that are L-isomers, e.g., L-nucleosides. Modifications to the sugar group can also include replacement of the 4'-O with sulfur, an optionally substituted nitrogen, or a CH2 group. In some embodiments, the linkage between C1' and the nucleobase is in the α-configuration.

[0508] Sugar modifications can also include acyclic nucleotides in which a C-C bond between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R and R are independently H, halogen, OR, or alkyl; and R is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0509] In some embodiments, the sugar modification is selected from the group consisting of 2'-H, 2'-O-Me (2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl, 2'-O-CH2-(4'-C) (LNA), 2'-O-CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and gem 2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0510] It should be understood that when a particular nucleotide is linked to the next nucleotide through its 2' position, the sugar modifications described herein can be located at the 3' position of the sugar for that particular nucleotide, e.g., the nucleotide linked through its 2' position. The modification at the 3' position can be in the xylose configuration. The term "xylose configuration" refers to the placement of the substituent on the C3' of the ribose in the same configuration as the 3'-OH in a xylose sugar.

[0511] The hydrogen attached to C4' and / or C1' can be replaced with a straight or branched chain, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein the backbone of the alkyl, alkenyl, and alkynyl can contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), a phosphorus-containing bond, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, wherein R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR 11 , C.O.R. 11 , CO2R 11 , [ka] , N.R. 21 R 31 ,CONR 21 R 31 , CON(H)NR 21 R 31 , ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 , OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 , ON=CR 41 R51 , SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; R 21 and R 31 is independently at each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 , or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is independently at each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , or CO2R 11 , or NR 11 R 11 ' and ;R 11 and R 11 is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is substituted.

[0512] In some embodiments, C4' and C5' together preferably form an optionally substituted heterocycle containing at least one -PX(Y)-, where X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino, where M is, independently for each occurrence, an alkylmetal or transition metal having a total charge of +1; and Y is O, S, or NR', where R' is hydrogen or an optionally substituted aliphatic. Preferably, this modification is at the 5' end of the iRNA.

[0513] In certain embodiments, the LNA has the formula: [ka] and a bicyclic nucleoside having the formula: During the ceremony: Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0514] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0515] In certain such embodiments, each of the substituted groups is independently mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, where each J1, J2, and J3 is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ1.

[0516] In certain embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), substituted alkoxy, or azido.

[0517] In certain embodiments, the Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In other embodiments, the Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0518] In certain such embodiments, the Z group is in the (R)-configuration: [ka] is located.

[0519] In certain such embodiments, the Z group is in the (S)-configuration: [ka] is located.

[0520] In certain embodiments, each of T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and dimethoxytrityl, where a more preferred hydroxyl protecting group is T1 which is 4,4'-dimethoxytrityl.

[0521] In certain embodiments, T2 is a reactive phosphorus group, and preferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In certain embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxyphosphoramidite.

[0522] In certain embodiments, the compounds of the present invention have the formula: [ka] or the expression: [ka] or the expression: [ka] and at least one monomer of During the ceremony, Bx is a heterocyclic base moiety; T3 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; T4 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amido.

[0523] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1.

[0524] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O or NJ1.

[0525] In certain such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, at least one Z is C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C1-C6 alkyl. In certain embodiments, each Z is independently substituted C1-C6 alkyl. In certain embodiments, at least one Z is substituted methyl. In certain embodiments, each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl.

[0526] In certain embodiments, at least one substituent is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In other embodiments, each substituent is independently C1-C6 alkoxy (e.g., each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxy).

[0527] In certain embodiments, at least one C1-C6 alkoxy substituent is CHO- (e.g., at least one Z is CHOCH-). In other embodiments, each C1-C6 alkoxy substituent is CHO- (e.g., each Z is CHOCH-).

[0528] In certain embodiments, at least one substituent is halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogens). In certain embodiments, each substituent is independently halogen (e.g., each Z is independently C1-C6 alkyl substituted with one or more halogens). In certain embodiments, at least one halogen substituent is fluoro (e.g., at least one Z is CH2FCH2-, CHF2CH2-, or CF3CH2-). In certain embodiments, each halo substituent is fluoro (e.g., each Z is independently CH2FCH2-, CHF2CH2-, or CF3CH2-).

[0529] In certain embodiments, at least one substituent is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, each substituent is independently hydroxyl (e.g., each Z is independently C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0530] In certain embodiments, at least one Z is CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-. In certain embodiments, each Z is independently CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-.

[0531] In certain embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In other embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0532] In certain embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0533] In certain embodiments, at least one Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; where each J1, J2, and J3 is independently H or C1-C6 alkyl and X is O, S, or NJ1. In certain embodiments, at least one Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0534] In certain embodiments, each Z group is independently -CH2Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O, S, or NJ1. In another embodiment, each Z group is independently -CH2Xx, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0535] In certain embodiments, at least one Z is CH3-. In other embodiments, each Z is CH3-.

[0536] In certain embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] The compound is in the (R)-configuration represented by

[0537] In certain embodiments, the Z group of each monomer of the formula is in the (R)-configuration.

[0538] In certain embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] The ion beam is in the (S)-configuration represented by

[0539] In certain embodiments, the Z group of each monomer of the formula is in the (S)-configuration.

[0540] In certain embodiments, T3 is H or a hydroxyl protecting group. In certain embodiments, T4 is H or a hydroxyl protecting group. In further embodiments, T3 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In certain embodiments, T4 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In certain embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In certain embodiments, T4 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In certain embodiments, T3 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, T4 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, at least one of T3 and T4 comprises an internucleoside linking group selected from phosphodiester or phosphorothioate.

[0541] In certain embodiments, the double-stranded iRNA agent of the invention has the formula: [ka] or the expression: [ka] or the expression: [ka] and at least one region of at least two consecutive monomers of

[0542] In certain such embodiments, LNAs include, but are not limited to, (A) α-L-methyleneoxy (4'-CH2-O-2') LNA, (B) β-D-methyleneoxy (4'-CH2-O-2') LNA, (C) ethyleneoxy (4'-(CH2)2-O-2') LNA, (D) aminooxy (4'-CH2-ON(R)-2') LNA, and (E) oxyamino (4'-CH2-N(R)-O-2') LNA, as shown below. [ka]

[0543] In certain embodiments, a double-stranded iRNA agent of the invention comprises at least two regions of at least two consecutive monomers of the above formula. In certain embodiments, a double-stranded iRNA agent of the invention comprises a gap motif. In certain embodiments, a double-stranded iRNA agent of the invention comprises at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, a double-stranded iRNA agent of the invention comprises at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0544] In certain embodiments, the double-stranded iRNA agent of the invention has the formula: [ka] wherein Bx is a heterocyclic base moiety. and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (S)-cEt monomer.

[0545] In certain embodiments, the monomer comprises a sugar mimetic. In certain such embodiments, the mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while the nucleobase is maintained for hybridization with a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some examples, the mimetics are used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those skilled in the art.

[0546] Nucleic acid modification (sugar bond) Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together to form oligomeric compounds, e.g., oligonucleotides. Such linking groups are also referred to as intersugar linkages. Two major classes of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to natural phosphodiester bonds, modified linkages can be used to alter, typica...

Claims

[Claim 1] 1. A method for reducing or inhibiting expression of a target gene in a subject, comprising: to said subject in need thereof, a) a double-stranded iRNA agent that includes: an antisense strand complementary to the target gene; a sense strand complementary to the antisense strand; 2'-OMe modifications to more than 15, more than 20, more than 25, or more than 30 nucleotides; and a carbohydrate-based ligand conjugated to at least one of said chains, optionally via a linker or carrier; and b) Penetration enhancers orally administering a formulation comprising:

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