Compositions and methods for treating or preventing Stargardt's disease and / or retinal binding protein 4 (RBP4)-associated disorders

Abstract

The present invention relates to agents that inhibit the expression and / or activity of transthyretin (TTR), such as double-stranded RNA (dsRNA) agents, or salts thereof, or antisense oligonucleotides, or gene therapy targeted to TTR, and the use of these agents in methods of treating or preventing Stargardt's disease, reducing vitamin A levels or the formation of toxic vitamin A metabolites, and / or halting the progression of vision loss in a subject. The present invention also relates to RNAi agents, such as double-stranded RNA (dsRNA) agents, targeting the retinal binding protein 4 (RBP4) gene.The present invention also relates to a method of using such RNAi agents to inhibit the expression of the RBP4 gene.The present invention further provides the use of RNAi agents targeting RBP4 and / or nucleic acid agents targeting TTR in methods of preventing and treating RBP4-related diseases, such as eye diseases, such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), such as dry AMD and wet AMD, or metabolic disorders, such as disorders of glucose and lipid homeostasis, such as insulin resistance associated with type II diabetes, or cardiovascular diseases.

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 208,027, filed June 8, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Stargardt disease is a genetic eye disorder that causes retinal degeneration and vision loss. Stargardt disease is a form of macular degeneration, also known as juvenile macular degeneration or "Stargardt macular degeneration." Stargardt disease is the most common form of hereditary macular degeneration, affecting approximately 30,000 people in the United States. The progressive vision loss associated with Stargardt disease is caused by degeneration of photoreceptor cells in the central part of the retina, called the macula. The macula is responsible for sharp central vision, which is required for detailed tasks such as reading, driving, and recognizing faces. In most people with Stargardt disease, a fatty yellow pigment (lipofuscin) accumulates in cells beneath the macula. Over time, the abnormal accumulation of this substance damages cells critical for clear central vision. In addition to central vision loss, people with Stargardt disease also experience night vision problems, which can make driving in low light difficult. Some affected individuals also have impaired color vision. The signs and symptoms of Stargardt disease typically appear in late childhood or early adulthood, worsen over time, and there is currently no cure. All that can be done is slow its progression with vitamin A supplements, avoidance of bright light, especially blue light, visual rehabilitation therapy, and the use of visual aids.

[0003] In subjects with Stargardt disease, lipofuscin accumulates in the RPE, the cells underlying the macula. These lipid-protein-retinoid aggregates are the primary cytotoxic components of RPE lipofuscin. Bisretinoids are vitamin A metabolites formed by the nonenzymatic reaction of vitamin A aldehyde in photoreceptor cells. Lipofuscin synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE, and the formation of a tertiary RBP4 / TTR / retinol complex in serum has been shown to be necessary for this influx.

[0004] Based on these findings, we hypothesized that reducing vitamin A levels and inhibiting lipofuscin formation by disrupting vitamin A transport to the eye through inhibition of RBP4 and / or TTR may be a viable treatment for subjects with Stargardt disease.

[0005] Indeed, Racz et al. (J Biol Chem. (2018) 293(29):11574-11588) demonstrated that administration of a non-retinoid RBP4 antagonist to the Abca4- / - knockout mouse model, an art-recognized mouse model of Stargardt disease, significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis.

[0006] However, mice carrying a targeted disruption of TTR (TTR-mice) had retinol and retinyl ester levels in the liver, testes, kidneys, spleen, and eyecups similar to those observed in wild-type mice, although they had less than 6% of the plasma retinol levels of wild-type mice. Furthermore, TTR-mice did not become blind and experienced severe weight loss, as seen in wild-type mice with similar vitamin A deficiency; rather, TTR-mice were phenotypically normal and fertile, and had the same lifespan as wild-type mice (Wei, et al. (1995) J. Biol. Chem. 273(2):866-870).

[0007] These differing results have caused confusion in the art and led to the expectation that reduction of vitamin A levels and treatment of Stargardt disease cannot be achieved by inhibiting the expression and activity of proteins involved in the transport of vitamin A, TTR, and RBP4 to the eye.

[0008] Thus, there is a need in the art for alternative treatments for subjects with Stargardt disease and / or RBP4-associated disorders, such as ocular diseases, metabolic diseases, e.g., disorders of glucose and lipid homeostasis, or cardiovascular diseases. Summary of the Invention

[0009] The present invention is based, at least in part, on the discovery that TTR knockout mice do not have reduced levels of vitamin A in the liver, testes, kidneys, spleen, and eyecups, but that administration of an agent that inhibits TTR expression, e.g., a nucleic acid agent targeting TTR, e.g., a double-stranded RNA (dsRNA) agent, e.g., butrisiran, surprisingly reduces vitamin A delivery to the eye via serum in a manner that can be used to treat conditions of intraocular hypervitaminosis A. It has also been discovered that serum vitamin A levels and TTR levels are highly correlated. Furthermore, it has been surprisingly discovered that despite a significant reduction in vitamin A levels, animals treated with an agent that inhibits TTR expression, e.g., a nucleic acid agent targeting TTR, e.g., a dsRNA agent, did not have impaired functional vision for at least 12 weeks, indicating that the vision of the treated animals was well preserved. Because diseases such as Stargardt syndrome are caused by excess toxic vitamin A metabolites, approaches that safely reduce vitamin A delivery to the eye without inducing visual defects from vitamin A deficiency offer novel opportunities for intervention. Thus, agents that inhibit the expression of TTR, e.g., nucleic acid agents that target TTR, e.g., double-stranded RNA (dsRNA) agents, e.g., butrisiran, can be used to treat subjects with Stargardt's disease.

[0010] The present invention is also based, at least in part, on the discovery of iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the gene encoding retinal binding protein 4 (RBP4). The RBP4 gene can be located within a cell, e.g., within a cell of a subject, such as a human subject. The present invention also provides methods of using the iRNA compositions of the present invention to inhibit expression of the RBP4 gene and / or to treat subjects who would benefit from inhibition or reduction of RBP4 gene expression, e.g., subjects suffering from or susceptible to an RBP4-associated disorder, e.g., ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, or metabolic disorders, e.g., disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, or cardiovascular disease.

[0011] Accordingly, in one aspect, the invention provides a method of treating or preventing at least one symptom in a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of an agent that inhibits expression and / or activity of transthyretin (TTR), thereby treating or preventing at least one symptom in a subject suffering from or susceptible to Stargardt disease.

[0012] In one aspect, the invention relates to a method for reducing vitamin A levels in the eye of a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of an agent that inhibits the expression and / or activity of transthyretin (TTR), thereby reducing vitamin A levels in the eye of the subject suffering from or susceptible to Stargardt disease.

[0013] In one aspect, the invention relates to a method for reducing the formation of toxic vitamin A metabolites in the retina of a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of an agent that inhibits the expression and / or activity of transthyretin (TTR), thereby reducing the formation of toxic vitamin A metabolites in the retina of the subject suffering from or susceptible to Stargardt disease.

[0014] In one aspect, the invention relates to a method of halting the progression of vision loss in a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of an agent that inhibits the expression and / or activity of transthyretin (TTR), thereby halting the progression of vision loss in an eye of the subject suffering from or susceptible to Stargardt disease.

[0015] In some embodiments, the agent that inhibits TTR expression and / or activity is selected from the group consisting of a small molecule inhibitor of TTR, a nucleic acid agent that targets TTR, and an anti-TTR antibody.

[0016] In some embodiments, the subject is a human.

[0017] In some embodiments, the agent is administered to the subject chronically.

[0018] In some embodiments, the agent is administered to the subject via subcutaneous, intramuscular, intravenous, or intravitreal administration.

[0019] In some embodiments, the agent is administered to the subject via subcutaneous administration. In some embodiments, the subcutaneous administration is self-administration. In some embodiments, the self-administration is via a pre-filled syringe or an auto-injector syringe.

[0020] In some embodiments, the agent is administered to the subject as a weight-based dose.

[0021] In some embodiments, the agent is administered to the subject as a fixed dose.

[0022] In some embodiments, the nucleic acid agent targeting TTR is a double-stranded RNA (dsRNA) agent, or a salt thereof, or an antisense oligonucleotide, or a gene therapy, that targets TTR.

[0023] In some embodiments, the nucleic acid agent is a dsRNA agent, or a salt thereof, that includes a sense strand that includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 20), and an antisense strand that includes the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 21), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: [ka] In the formula, X is O.

[0024] In some embodiments, the dsRNA agent, or a salt thereof, is administered to the subject at a dose of about 25 mg. In some embodiments, the dsRNA agent, or a salt thereof, is administered to the subject approximately once every three months. In some embodiments, the dsRNA agent, or a salt thereof, is administered subcutaneously to the subject. In some embodiments, the dsRNA agent, or a salt thereof, is present in a pharmaceutical composition. In some embodiments, the dsRNA agent is in salt form. In some embodiments, the dsRNA agent, or a salt thereof, is administered to the subject at a dose of about 25 mg approximately once every three months.

[0025] In some embodiments, the nucleic acid agent is a dsRNA agent, or a salt thereof, comprising a sense strand comprising the nucleotide sequence 5'-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAf-3' (SEQ ID NO: 22), and an antisense strand comprising the nucleotide sequence 5'-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3' (SEQ ID NO: 23), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: [ka] In the formula, X is O.

[0026] In some embodiments, the dsRNA agent, or salt thereof, is administered to the subject at a dose of about 50-500 mg. In some embodiments, the dsRNA agent, or salt thereof, is administered to the subject once a week. In some embodiments, the dsRNA agent, or salt thereof, is administered subcutaneously to the subject. In some embodiments, the dsRNA agent is present in a pharmaceutical composition. In some embodiments, the dsRNA agent is in salt form. In some embodiments, the dsRNA agent is administered to the subject at a dose of 500 mg once daily for five days, followed by a dose of 500 mg once a week.

[0027] In some embodiments, the nucleic acid agent is a dsRNA agent comprising a sense strand that comprises the nucleotide sequence 5'-GuAAccAAGAGuAuuccAudTdT-3' (SEQ ID NO: 24) and an antisense strand that comprises the nucleotide sequence 5'-AUGGAAuACUCUUGGUuACdTdT-3' (SEQ ID NO: 25), or a salt thereof, where A is adenosine, C is cytidine, G is guanosine, U is uridine, a is 2'-O-methyladenosine, c is 2'-O-methylcytidine, g is 2'-O-methylguanosine, u is 2'-O-methyluridine, and dT is 2'-deoxythymidine.

[0028] In some embodiments, the subject weighs less than about 100 kg and is administered a dose of about 0.3 mg / kg of the dsRNA agent, or a salt thereof. In some embodiments, the subject weighs more than about 100 kg and is administered a dose of about 30 mg / kg of the dsRNA agent, or a salt thereof.

[0029] In some embodiments, the dsRNA agent, or a salt thereof, is administered to a subject once every three weeks. In some embodiments, the dsRNA agent, or a salt thereof, is administered to a subject via intravenous infusion. In some embodiments, the dsRNA agent is present in a pharmaceutical composition. In some embodiments, the dsRNA agent is in salt form. In some embodiments, the dsRNA agent, or a salt thereof, is administered at a dose of about 0.3 mg / kg of the dsRNA agent, or a salt thereof, once every three weeks to a subject weighing less than about 100 kg, or at a dose of about 30 mg / kg of the dsRNA agent, or a salt thereof, once every three weeks to a subject weighing more than about 100 kg.

[0030] In some embodiments, the nucleic acid agent is a single-stranded modified oligonucleotide of 20 linked nucleosides having a nucleobase sequence of 5'-TCTTGGTTACATGAAATCCC-3' (SEQ ID NO: 26), wherein the modified oligonucleotide comprises a gap segment of 10 linked deoxynucleosides, a 5' wing segment of 5 linked nucleosides, and a 3' wing segment of 5 linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage, and each cytosine in the modified oligonucleotide is a 5-methylcytosine.

[0031] In some embodiments, the single-stranded modified oligonucleotide is administered to the subject at a fixed dose of about 284 mg. In some embodiments, the single-stranded modified oligonucleotide is administered to the subject about once a week. In some embodiments, the single-stranded modified oligonucleotide is administered subcutaneously to the subject. In some embodiments, the single-stranded modified oligonucleotide is present in a pharmaceutical composition. In some embodiments, the single-stranded modified oligonucleotide is administered to the subject at a dose of about 284 mg once a week.

[0032] In one aspect, the invention provides a method of treating or preventing at least one symptom of Stargardt disease in a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of a dsRNA agent, or a salt thereof, comprising: a sense strand comprising the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 20), and an antisense strand comprising the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 21), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and a ligand is conjugated to the 3' end of the sense strand, as shown in the following schematic diagram: [ka] wherein X is O, thereby treating or preventing at least one symptom in a subject suffering from or susceptible to Stargardt's disease.

[0033] In another aspect, the invention provides a method for reducing vitamin A levels in the eye of a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of a dsRNA agent, or a salt thereof, comprising: a sense strand comprising the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 20), and an antisense strand comprising the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 21), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and a ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: [ka] wherein X is O, thereby decreasing vitamin A levels in the eye of a subject suffering from or susceptible to Stargardt's disease.

[0034] In another aspect, the invention provides a method for reducing the formation of toxic vitamin A metabolites in the retina of a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of a dsRNA agent, or a salt thereof, comprising: a sense strand comprising the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO:20), and an antisense strand comprising the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO:21), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and a ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: [ka] wherein X is O, thereby reducing the formation of toxic vitamin A metabolites in the retina of subjects suffering from or susceptible to Stargardt's disease.

[0035] In another aspect, the invention provides a method of halting the progression of vision loss in a subject suffering from or susceptible to Stargardt disease, the method comprising administering to the subject an effective amount of a dsRNA agent, or a salt thereof, comprising: a sense strand comprising the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 20), and an antisense strand comprising the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 21), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and a ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: [ka] wherein X is O, thereby halting the progression of vision loss in the eye of a subject suffering from or susceptible to Stargardt's disease.

[0036] In some embodiments, the dsRNA agent, or its salt, is administered to the subject at a dose of about 25 mg. In some embodiments, the dsRNA agent, or its salt, is administered to the subject approximately once every three months. In some embodiments, the dsRNA agent, or its salt, is administered subcutaneously to the subject. In some embodiments, the dsRNA agent, or its salt, is present in a pharmaceutical composition. In some embodiments, the dsRNA agent is in a salt form.

[0037] In some embodiments, the dsRNA agent, or salt thereof, is administered to the subject as a dose of about 25 mg about once every three months.

[0038] In some embodiments, administration of the agent to a subject reduces fundus autofluorescence as determined by Fundus Autoflourescence Photography (FAF).

[0039] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent for the treatment of Stargardt's disease.

[0040] In some embodiments, the additional therapeutic agent is selected from the group consisting of agents that inhibit the expression and / or activity of transthyretin (TTR), the synthetic retinoid fenretinide, anti-VEGF therapy, corticosteroids, insulin, glucagon-like peptide 1 agonists, sulfonylureas, seglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, and combinations of any of the foregoing.

[0041] In some embodiments, the method further comprises determining the level of RBP4 and / or TTR in a sample from the subject.

[0042] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of retinal binding protein 4 (RBP4) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 0, 1, 2, or 3 nucleotides, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 1, 2, or 3 nucleotides.

[0043] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of retinal binding protein 4 (RBP4) in a cell, the dsRNA comprising a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding RBP4, and the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ by 0, 1, 2, or 3 or less nucleotides from any one of the antisense nucleotide sequences of any one of Tables 2-3.

[0044] In one embodiment, the dsRNA agent includes at least one modified nucleotide.

[0045] In one embodiment, substantially all of the nucleotides in the sense strand, substantially all of the nucleotides in the antisense strand contain a modification, or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand contain a modification.

[0046] In one embodiment, all of the nucleotides in the sense strand comprise a modification, all of the nucleotides in the antisense strand comprise a modification, or all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0047] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythimidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl ... The nucleotides are selected from the group consisting of modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, thermolabile nucleotides, glycol modified nucleotides (GNAs), nucleotides containing 2' phosphates, and 2-O-(N-methylacetamido) modified nucleotides, and combinations thereof.

[0048] In one embodiment, the modification to the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol, and combinations thereof.

[0049] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, glycol-modified nucleotides (GNAs), such as Ggn, Cgn, Tgn, or Agn, nucleotides having a 2' phosphate, such as G2p, C2p, A2p, U2p, and vinyl-phosphonate nucleotides, and combinations thereof.

[0050] In another embodiment, at least one of the modifications to the nucleotides is a thermally destabilizing nucleotide modification.

[0051] In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposing nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0052] In some embodiments, the modified nucleotides include a short sequence of 3'-terminal deoxythymidine nucleotides (dT).

[0053] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5'-end and the 3'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand.

[0054] The double-stranded region can be 19 to 30 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0055] In one embodiment, each strand is independently 30 nucleotides or less in length.

[0056] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0057] The region of complementarity can be at least 17 nucleotides in length, 19-23 nucleotides in length, or 19 nucleotides in length.

[0058] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0059] In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0060] In one embodiment, the dsRNA agent further comprises a ligand.

[0061] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0062] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0063] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0064] In one embodiment, the ligand is: [ka] .

[0065] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] wherein X is O or S.

[0066] In one embodiment, X is O.

[0067] In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0068] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand, for example, the antisense strand or the sense strand.

[0069] In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand, for example, the antisense strand or the sense strand.

[0070] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand, hi one embodiment, the strand is the antisense strand.

[0071] In one embodiment, the base pair at one position at the 5'-end of the antisense strand of the duplex is an AU base pair.

[0072] The invention also provides cells containing any of the dsRNA agents of the invention, and pharmaceutical compositions comprising any of the dsRNA agents of the invention.

[0073] A pharmaceutical composition of the invention can include a dsRNA agent in an unbuffered solution, e.g., saline or water, or a pharmaceutical composition of the invention can include a dsRNA agent in a buffered solution, e.g., a buffered solution containing acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof, or phosphate buffered saline (PBS).

[0074] In one aspect, the present invention provides a method for inhibiting expression of the retinal binding protein 4 (RBP4) gene in a cell, comprising contacting the cell with any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby inhibiting expression of the RBP4 gene in the cell.

[0075] In another aspect, the present invention provides a method for inhibiting the expression and / or activity of the retinal binding protein 4 (RBP4) gene in a cell, comprising contacting the cell with an agent that inhibits the expression and / or activity of TTR, e.g., a small molecule or nucleic acid agent that targets transthyretin (TTR), e.g., an siRNA or antisense oligonucleotide that targets TTR, or gene therapy, thereby inhibiting the expression of the RBP4 gene in the cell.

[0076] In one embodiment, the cell is in a subject, e.g., a human subject, e.g., a subject having a retinal binding protein 4 (RBP4)-associated disorder, e.g., an ocular disease, e.g., Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, or a metabolic disorder, e.g., a disorder of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, or cardiovascular disease.

[0077] In certain embodiments, the RBP4-associated disorder is Stargardt's disease. In certain embodiments, the RBP4-associated disorder is diabetic retinopathy. In certain embodiments, the RBP4-associated disorder is age-related macular degeneration (AMD), for example, dry AMD or wet AMD.

[0078] In certain embodiments, the RBP4-associated disorder is insulin resistance associated with type II diabetes.

[0079] In certain embodiments, RBP4 expression is inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one embodiment, inhibiting expression of RBP4 reduces RBP4 protein levels in the subject's serum by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0080] In certain embodiments, TTR expression and / or activity levels are inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In certain embodiments, TTR activity levels, e.g., retinol binding to TTR and / or RBP4, retinol / RBP4 / TTR complex formation, or retinol transport or delivery to target tissues, are inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0081] In one aspect, the present invention provides a method for treating a subject having a disorder that would benefit from reduced retinal binding protein 4 (RBP4) expression. The method comprises administering to the subject a therapeutically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby treating the subject having a disorder that would benefit from reduced RBP4 expression.

[0082] In another aspect, the present invention provides a method for preventing at least one symptom in a subject with a disorder that would benefit from reduced retinal binding protein 4 (RBP4) expression. The method comprises administering to the subject a prophylactically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby preventing at least one symptom in a subject with a disorder that would benefit from reduced RBP4 expression.

[0083] In one aspect, the present invention provides a method of treating a subject having a disorder that would benefit from reduced retinal binding protein 4 (RBP4) expression. The method includes administering to the subject a therapeutically effective amount of an agent that inhibits TTR expression and / or activity, e.g., a small molecule or nucleic acid agent that targets transthyretin (TTR), e.g., a TTR-targeting siRNA or antisense oligonucleotide, or gene therapy, thereby treating the subject having a disorder that would benefit from reduced RBP4 expression.

[0084] In another aspect, the present invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced retinal binding protein 4 (RBP4) expression. The method includes administering to the subject a prophylactically effective amount of an agent that inhibits TTR expression and / or activity, e.g., a small molecule or nucleic acid agent that targets transthyretin (TTR), e.g., a TTR-targeting siRNA or antisense oligonucleotide, or gene therapy, thereby preventing at least one symptom in the subject having the disorder that would benefit from reduced RBP4 expression.

[0085] In certain embodiments, the disorder is a retinal binding protein 4 (RBP4)-associated disorder, e.g., an ocular disease, e.g., Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, or a metabolic disorder, e.g., a disorder of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, or a cardiovascular disease.

[0086] In some embodiments, the RBP4-associated disorder is Stargardt's disease. In some embodiments, the RBP4-associated disorder is age-related macular degeneration, e.g., dry AMD or wet AMD. In some embodiments, the RBP4-associated disorder is diabetic retinopathy. In some embodiments, the RBP4-associated disorder is insulin resistance associated with type II diabetes.

[0087] In certain embodiments, administration of the dsRNA to a subject causes a decrease in the accumulation of RBP4 and / or TTR protein in the subject.

[0088] In certain embodiments, administration of the dsRNA to a subject reduces the accumulation of lipofuscin pigment in the eye. In certain embodiments, administration of the dsRNA to a subject causes a decrease in neovascularization and / or a decrease in drusen accumulation in the eye.

[0089] In certain embodiments, administering the dsRNA to a subject increases insulin sensitivity in the subject.

[0090] In a further aspect, the present invention also provides a method for inhibiting expression of RBP4 in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the dsRNAs provided herein, thereby inhibiting expression of RBP4 in the subject.

[0091] In one embodiment, the subject is a human.

[0092] In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0093] In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

[0094] In certain embodiments, the dsRNA agent is administered to the subject via periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, anterior or posterior juxtascleral, subretinal, subconjunctival, retrobulbar, or intracanalicular administration.

[0095] In one embodiment, the dsRNA agent is administered to the subject intravitreally.

[0096] In one embodiment, the methods of the invention further comprise determining the level of RBP4 and / or TTR in a sample from the subject.

[0097] In one embodiment, the level of RBP4 and / or TTR in a subject sample is the level of RBP4 and / or TTR protein in blood or serum, or an eye or liver tissue sample.

[0098] In certain embodiments, the methods of the present invention further comprise administering an additional therapeutic agent to the subject.

[0099] In certain embodiments, the additional therapeutic agent is selected from the group consisting of an agent that inhibits TTR expression and / or activity, e.g., a small molecule or nucleic acid agent that targets TTR, e.g., a siRNA, or antisense oligonucleotide, or gene therapy that targets TTR, a TTR small molecule inhibitor, or an anti-TTR antibody; an anti-VEGF therapy, a corticosteroid, insulin, a glucagon-like peptide 1 agonist, a sulfonylurea, a segritinide, a biguanide, a thiazolidinedione, an alpha-glucosidase inhibitor, an SGLT2 inhibitor, a DPP-4 inhibitor, an HMG-CoA reductase inhibitor, and a combination of any of the foregoing.

[0100] The present invention also provides a kit comprising any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, and optionally instructions for use. In one embodiment, the present invention provides a kit for carrying out a method of inhibiting expression of the RBP4 gene in a cell by contacting the cell with a double-stranded RNAi agent of the present invention in an amount effective to inhibit expression of RBP4 in the cell. The kit comprises the RNAi agent, instructions for use, and optionally a means for administering the RNAi agent to a subject. [Brief explanation of the drawings]

[0101] [Figure 1]Graphs showing the mean percent change from baseline in serum vitamin A concentration over time (top graph) and the mean percent change from baseline in serum TTR concentration over time (bottom graph) in subjects who received a single subcutaneous dose of the indicated dose of butrisiran (5 mg, 25 mg, 50 mg, 100 mg, 200 mg, or 300 mg). Baseline values ​​are defined as the average of all measurements taken before administration of butrisiran. [Figure 2] Graphs showing the mean percent change in serum vitamin A levels in subjects receiving 25 mg of butrisiran subcutaneously every three months or 0.3 mg / kg of patisiran intravenously every three months over an 18-month treatment period (top graph), and the correlation between serum vitamin A levels and serum TTR levels in these subjects (bottom graph). [Figure 3] 1 is a graph showing the correlation between observed (dashed line) and modeled (solid line) changes in baseline vitamin A levels over time in subjects receiving a single 25 mg dose of butrisiran subcutaneously every three months. The shaded area is the 90% prediction interval from the simulation. [Figure 4] 1 is a graph and table showing the predicted decrease in serum vitamin A levels from baseline in subjects receiving 25 mg of butrisiran subcutaneously every three months over a 96-week period. The line represents the median and the shaded area represents the 90% prediction interval from the simulation. [Figure 5] Figure 1 shows predicted decreases in vitamin A and TTR levels in adult and adolescent subjects receiving 25 mg of butrisiran subcutaneously every 3 months over a 96-week period. The line represents the median, and the shaded area represents the 90% prediction interval from the simulation. Adult weight: 78.6 (39.1, 231) kg; 12-17 year old weight: 61.7 (29.1, 150) kg. [Figure 6A]1 is a graph showing the mean percent change in liver TTR mRNA levels in wild-type (WT) mice and Abca4- / - Rdh8- / - double knockout (DKO) mice administered a 0.3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously once every 3 weeks for 12 weeks. [Figure 6B] 1 is a graph showing serum TTR protein persistence compared to pre-dose levels at baseline and on days 21, 42, 63, and 84 of the study in wild-type (WT) and Abca4- / - Rdh8- / - double knockout (DKO) mice administered a 3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously every 3 weeks for 12 weeks. [Figure 7A] 1 is a graph showing serum RPB4 protein persistence compared to pre-dose levels at baseline and on days 21, 42, 63, and 84 of the study in wild-type (WT) and Abca4- / - Rdh8- / - double knockout (DKO) mice administered a 3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously every 3 weeks for 12 weeks. [Figure 7B] 1 is a graph showing serum retinol levels in wild-type (WT) and Abca4- / - Rdh8- / - double knockout (DKO) mice administered a 3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously every 3 weeks for 12 weeks at baseline and on days 21, 42, 63, and 84 of the study. [Figure 8] 1 is a graph showing the correlation between serum retinol levels and serum RBP4 levels in wild-type (WT) mice and Abca4- / - Rdh8- / - double knockout (DKO) mice administered a 3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously once every 3 weeks for 12 weeks. [Figure 9A]

[0023] Figure 1 shows exemplary two-photon images of the retinal pigment epithelium (RPE) at baseline and 12 weeks in Abca4- / - Rdh8- / - double knockout (DKO) mice administered a 3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously every 3 weeks for 12 weeks. The top row of images shows fluorescence at 730 nm, and the bottom row of images shows fluorescence at 850 nm. [Figure 9B] 9B is a graph showing the fluorescence ratio 850 nm / 730 nm in the microscopic image of FIG. 9A. [Figure 10] 1 is a graph showing the scotopic ERG responses of DKO mice administered a 3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously every 3 weeks for 12 weeks at baseline and at week 12 of the study. [Figure 11] 1 is a graph showing photopic ERG responses of DKO mice administered a 3 mg / kg dose of AD-64958, a dsRNA agent targeting TTR, subcutaneously every 3 weeks for 12 weeks at baseline and at week 12 of the study. DETAILED DESCRIPTION OF THE INVENTION

[0102] The present invention is based, at least in part, on the discovery that TTR knockout mice do not have reduced levels of vitamin A in the liver, testes, kidneys, spleen, and eyecups, but that administration, e.g., systemic administration, of an agent that inhibits TTR expression, e.g., a nucleic acid agent targeting TTR, e.g., a double-stranded RNA (dsRNA) agent, e.g., butrisiran, surprisingly reduces vitamin A delivery to the eye via serum in a manner that can be used to treat conditions of intraocular hypervitaminosis A. It has also been discovered that, for example, serum vitamin A levels and TTR levels are highly correlated. Furthermore, it has been surprisingly discovered that, despite a significant reduction in vitamin A levels, animals treated with an agent that inhibits TTR expression, e.g., a nucleic acid agent targeting TTR, e.g., a dsRNA agent, do not have impaired functional vision for at least 12 weeks, indicating that the vision of the treated animals is well preserved. Because diseases such as Stargardt disease are caused by excess toxic vitamin A metabolites, approaches that safely reduce vitamin A delivery to the eye without inducing visual defects from vitamin A deficiency offer novel opportunities for intervention. Thus, agents that inhibit TTR expression, such as nucleic acid agents that target TTR, e.g., double-stranded RNA (dsRNA) agents, such as butrisiran, can be used to treat subjects with Stargardt disease.

[0103] Accordingly, the present invention provides a method for treating a subject with Stargardt disease, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits the expression and / or activity of transthyretin (TTR), thereby treating the subject with Stargardt disease.

[0104] The present invention also provides methods for reducing vitamin A levels in the eye of a subject suffering from or susceptible to Stargardt disease, methods for reducing the formation of toxic vitamin A metabolites in the retina of a subject suffering from or susceptible to Stargardt disease, and methods for halting the progression of vision loss in a subject suffering from or susceptible to Stargardt disease, which methods comprise administering to the subject a therapeutically effective amount of an agent that inhibits the expression and / or activity of transthyretin (TTR).

[0105] Additionally, the present invention provides iRNA compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the retinal binding protein 4 (RBP4) gene. The gene can be located within a cell, e.g., within a subject, such as a human. Use of these iRNAs allows for targeted degradation of the mRNA of the corresponding gene (RBP4) in a mammal.

[0106] The iRNAs of the present invention are designed to target the human retinal binding protein 4 (RBP4) gene, including portions of the gene that are conserved in RBP4 orthologs of other mammalian species. Without intending to be limited by theory, it is believed that combinations or subcombinations of the above features and specific target sites or specific modifications in these iRNAs improve the efficacy, stability, potency, durability, and safety of the iRNAs of the present invention.

[0107] Thus, the present invention provides methods for treating and preventing retinal binding protein 4 (RBP4)-associated disorders, such as ocular diseases, e.g., Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, or metabolic disorders, e.g., disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, or cardiovascular disease, using iRNA compositions that result in RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the RBP4 gene.

[0108] The iRNA of the present invention comprises an RNA strand (antisense strand) having a region of up to about 30 nucleotides in length, for example, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the RBP4 gene.

[0109] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the RBP4 gene. In some embodiments, such iRNA agents with longer antisense strands can include a second RNA strand (sense strand) that is, for example, 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0110] The use of iRNAs of the present invention allows for the targeted degradation of mRNA of the corresponding gene (RBP4 gene) in mammals. Using in vitro assays, the inventors have demonstrated that iRNAs targeting the RBP4 gene can potently mediate RNAi, resulting in significant inhibition of RBP4 gene expression. Therefore, methods and compositions comprising these iRNAs are useful for treating subjects with RBP4-related disorders, such as eye diseases, such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), including dry AMD and wet AMD, or metabolic disorders, such as glucose and lipid homeostasis disorders, such as insulin resistance associated with type II diabetes, or cardiovascular disease.

[0111] The following detailed description of the invention discloses methods of making and using compositions containing iRNA that inhibit expression of the RBP4 gene, as well as compositions, uses, and methods of treating subjects who would benefit from inhibition and / or reduction of expression of the RBP4 gene, e.g., subjects susceptible to or diagnosed with an RBP4-associated disorder.

[0112] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Additionally, it should be noted that whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0113] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or to more than one element, e.g., a plurality of elements.

[0114] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0115] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. For example, "the sense strand or the antisense strand" is understood as "the sense strand or the antisense strand, or the sense strand and the antisense strand."

[0116] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or ranges, it is understood that "about" can modify each of the consecutive numbers or ranges.

[0117] The terms "at least," "greater than," or "or more" preceding a number or series of numbers are understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may be logically included, if this is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides of a 21-nucleotide nucleic acid molecule" means that 19, 20, or 21 nucleotides have the specified property. When at least precedes a series of numbers or ranges, it is understood that "at least" can modify each of the series of numbers or ranges.

[0118] As used herein, "less than" or "or less than" is understood to mean the value adjacent to the phrase and any logically smaller value or integer than that value, up to zero, where logical from the context. For example, a duplex having an overhang of "2 nucleotides or less" has 2, 1, or 0 nucleotide overhangs. When "less than" precedes a series of numbers or ranges, it is understood that the "less than" can modify each of the series of numbers or ranges. As used herein, a range includes both the upper and lower limits.

[0119] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0120] In the event of a conflict between the nucleotide sequence for a given target site and either the sense or antisense strand, the given sequence controls.

[0121] In the event of a discrepancy between a sequence on a transcript or other sequence and its indicated site, the nucleotide sequence listed herein takes precedence.

[0122] As used herein, the term "retinal binding protein 4," used interchangeably with "RBP4," refers to a member of the lipocalin family of major transport proteins for the hydrophobic molecule retinol, also known as vitamin A, in the circulation (Kanai, M., Raz, A., and Goodman, D.S. (1968). J. Clin. Invest. 47, 2025-2044). RBP4 is also known as plasma retinol-binding protein MCOPCB10, RDCCAS, PRBP, or RBP. RBP4 expression is highest in the liver, where the majority of the body's vitamin A reserves are stored as retinyl esters. For vitamin A mobilization from the liver, retinyl esters are hydrolyzed to retinol, which then binds to RBP4 in hepatocytes. After associating with transthyretin (TTR), the retinol / RBP4 / TTR complex is released into the bloodstream and delivers retinol to tissues via binding to specific membrane receptors. Upon retinol release and RBP4 dissociation from TTR, the retinol-free circulating RBP4 is filtered by the kidney. More than 99% of it is reabsorbed by the proximal tubule, making urinary RBP4 a sensitive marker of renal tubular dysfunction (Bonventre, JV, et al., (2010). Nat. Biotechnol. 28, 436-440). RBP4 levels are further regulated by TTR.

[0123] Vitamin A deficiency is manifested by visual impairment and can lead to night blindness or complete blindness (Blegvad O. (1924). Am. J. Ophthalmol. 7, 89-117). Vitamin A deficiency due to malnutrition during pregnancy is the main cause of visual defects in newborns in developing countries (Pirie A, (1983) Proc. Nutr. Soc. 42, 53-64). Indeed, mice lacking RBP4 exhibit impaired retinal function and vision, and transgenic expression of human RBP4 either in muscle (Quadro L. et al., (2002). J. Biol. Chem. 277, 30191-30197) or from the mouse Rbp4 locus (Liu L. et al., (2017). Lab. Invest. 97, 395-408) rescued serum retinol levels and suppressed the visual defects caused by loss of endogenous RBP4. These results indicate that visual function depends on RBP4 / TTR-mediated retinol transport.

[0124] While most of the functions of RBP4 and its role in retinol transport and homeostasis have focused on vision and ocular diseases, the role of RBP4 has also been investigated in other diseases. For example, elevated circulating RBP4 in patients with type 2 diabetes was reported several years ago (Basualdo C, et al., (1997). J. Am. Coll. Nutr. 16, 39-45; Abahusain et al., (1999). Eur. J. Clin. Nutr. 53, 630-635), and transgenic overexpression of RBP4 or injection of human RBP4 in normal mice was shown to cause insulin resistance. In contrast, genetic deletion of RBP4 or reduction of circulating RBP4 levels had the opposite effect, protecting mice from the development of insulin resistance (Yang, Q., et al. (2005). Nature 436, 356-362). Furthermore, positive associations have been documented for circulating RBP4 or RBP4 expression levels with established cardiovascular disease (CVD) risk factors, including metabolic syndrome, systemic / central obesity, dyslipidemia, inflammatory markers, and hypertension (Qi Q, et al., J Clin Endocrinol Metab. 2007;92:4827-4834, Ingelsson E, et al., Atherosclerosis. 2009;206:239-244). Thus, RBP4 is involved in a variety of human conditions, including not only visual impairment and eye diseases, but also metabolic diseases such as impaired glucose and lipid homeostasis and cardiovascular disease (Li, Z. et al., (2010). J. Int. Med. Res. 38, 95-99; Yang, Q., et al. (2005). Nature 436, 356-362; Sun, Q., et al. (2013). Circulation 127, 1938-1947).

[0125] The sequence of human RBP4 mRNA transcript can be found, for example, in GenBank accession number GI:1519313037 (NM_006744.4, SEQ ID NO:1, reverse complement, SEQ ID NO:2). The sequence of mouse RBP4 mRNA can be found, for example, in GenBank accession number GI:226958687 (NM_001159487.1, SEQ ID NO:3, reverse complement, SEQ ID NO:4). The sequence of rat RBP4 mRNA can be found, for example, in GenBank accession number GI:158187534 (NM_013162.1, SEQ ID NO:5, reverse complement, SEQ ID NO:6). The sequence of cynomolgus monkey RBP4 mRNA can be found, for example, in GenBank accession number GI:982269650 (XM_005565974.2, SEQ ID NO:7, reverse complement, SEQ ID NO:8). The sequence of rhesus monkey RBP4 mRNA can be found, for example, in GenBank Accession No. GI:1622966559 (XM_015147709.2, SEQ ID NO: 9, reverse complement, SEQ ID NO: 10).

[0126] Further examples of RBP4 mRNA sequences are readily available through public databases such as, for example, GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0127] Further information regarding RBP4 can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=RBP4.

[0128] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0129] The term RBP4, as used herein, also refers to variations of the RBP4 gene, including variants provided in SNP databases. Numerous sequence variations within the RBP4 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, e.g., www.ncbi.nlm.nih.gov / snp / ?term=RBP4, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0130] As used herein, transthyretin (TTR), also known as prealbumin, ATTR, CTS, CTS1, HEL111, HsT2651, PALB, TBPA, and TTN, refers to a highly conserved protein that functions as a transporter of the thyroid hormone thyroxine (T4) and retinol (vitamin A)-bound retinol-binding protein (RBP). Specifically, TTR acts as a carrier of retinol (vitamin A) through its association with RBP in blood and CSF. TTR is a tetramer of four identical 127-amino acid subunits (monomers) rich in beta-sheet structure. Each monomer has two four-stranded beta-sheets and a prolate ellipsoidal shape. Antiparallel beta-sheet interactions connect the monomers into a dimer. A short loop from each monomer forms the main dimer-dimer interaction. Two pairs of these loops separate the opposing convex beta-sheets of the dimer to form an internal channel. The liver is the primary site of TTR expression, and most of the body's vitamin A reserves are stored as retinyl esters. Other important expression sites include the choroid plexus, retina (especially the retinal pigment epithelium), and pancreas. To mobilize vitamin A from the liver, retinyl esters are hydrolyzed to retinol, which can then bind to RBP4 in hepatocytes. TTR then binds to RBP4, forming a retinol / RBP4 / TTR complex, which is released into the bloodstream and delivers retinol to tissues throughout the body via binding to specific membrane receptors. When retinol is released and RBP4 dissociates from TTR, the retinol-free RBP4 in circulation is filtered by the kidney. More than 99% of it is reabsorbed by the proximal tubule (Bonventre, JV, et al., (2010). Nat. Biotechnol. 28, 436-440).

[0131] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the RBP4 gene, including mRNA that is the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-dependent cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the RBP4 gene.

[0132] The target sequence can be about 19-36 nucleotides in length, e.g., about 19-30 nucleotides in length. For example, the target sequence can be about 19-30, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19 to 23 nucleotides in length, optionally 21 to 23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0133] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0134] Generally, "G", "C", "A", "T" and "U" respectively represent nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases.However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 1).Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially changing the base pairing properties of the oligonucleotide containing the nucleotide with such replacement moiety.For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil.Therefore, a nucleotide containing uracil, guanine or adenine can be substituted with a nucleotide containing inosine, for example, in the nucleotide sequence of the dsRNA of the present invention. In another embodiment, adenine and cytosine anywhere within an oligonucleotide can be substituted with guanine and uracil, respectively, to form GU wobble bases that pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured herein.

[0135] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, refer to agents that contain RNA as those terms are defined herein and mediate cleavage of targets of RNA transcription via the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate, e.g., inhibit, expression of the RBP4 gene in cells, e.g., cells in a subject, such as a mammalian subject.

[0136] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, for example, an RBP4 target mRNA sequence, and induces cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes this dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). siRNA is then incorporated into RNA-induced silencing complex (RISC), where one or more helicases can unwind the siRNA duplex, thereby inducing target recognition for complementary antisense strands (Nykanen, et al., (2001) Cell 107:309).When bound to appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev.15:188).That is, in one aspect, the present invention relates to the single-stranded RNA (siRNA) that is produced in cells and promotes the formation of RISC complex, resulting in the silencing of target gene, i.e., RBP4 gene.Therefore, the term "siRNA" is also used herein to refer to the iRNA described above.

[0137] In certain embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and chemically modified. The design and testing of single-stranded siRNAs is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs described herein or as single-stranded siRNAs chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0138] In certain embodiments, the "iRNA" used in the compositions, uses, and methods of the invention is double-stranded RNA, and is referred to herein as a "double-stranded RNA agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having a "sense" or "antisense" orientation with respect to the target RNA, i.e., the RBP4 gene. In some embodiments of the invention, the double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0139] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, "iRNA" may include ribonucleotides with chemical modifications, and iRNA may contain substantial modifications in multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, a functional group or atom, to the internucleoside bond, sugar moiety, or nucleobase. Modifications suitable for use in the agent of the present invention include all types of modifications disclosed herein or known in the art. When used in siRNA-type molecules, all such modifications are encompassed by "iRNA" or "RNAi agent" for the purposes of this specification and claims.

[0140] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides can be considered to constitute modified nucleotides when present within an RNAi agent.

[0141] The double-stranded region can be any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can be about 19 to 36 base pairs in length, e.g., about 19 to 30 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 19 to 30, 19 The double-stranded region may range in length from 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. In certain embodiments, the double-stranded region is 19 to 21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also contemplated as part of this disclosure.

[0142] The two strands forming a double-stranded structure can be different portions of a single larger RNA molecule, or they can be separate RNA molecules. When two strands are part of a single larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand that form the duplex structure, the connected RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 nucleotides.

[0143] When the two substantially complementary strands of a dsRNA are composed of separate RNA molecules, they may, but need not, be covalently linked. When the two strands are covalently linked by a means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the corresponding 5'-end of the other strand forming the double-stranded structure, the connecting structure is referred to as a "linker." The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the double-stranded structure, the RNAi may contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0144] In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, eg, the RBP4 gene, to induce cleavage of the target RNA.

[0145] In some embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, an RBP4 target mRNA sequence, and induces cleavage of the target RNA.

[0146] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide, or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. An overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, an overhanging nucleotide may be present on the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0147] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0148] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0149] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end or 5'-end, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can comprise an extended length of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides in length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is present on the 3'-end of the antisense strand of the double strand.In certain embodiments, the extended overhang is present on the 5'-end of the antisense strand of the double strand.In certain embodiments, one or more of the nucleotides in the extended overhang are replaced with nucleoside thiophosphate.In certain embodiments, the overhang comprises a self-complementary portion, so that the overhang can form a stable hairpin structure under physiological conditions.

[0150] "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at the ends of a double-stranded RNA agent, i.e., there are no nucleotide overhangs.A "blunt-ended" double-stranded RNA agent is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule.The RNAi agents of the present invention include RNAi agents that have no nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end), or RNAi agents that have no nucleotide overhangs at either end.In most cases, such molecules will be double-stranded throughout their entire length.

[0151] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., RBP4 mRNA.

[0152] As used herein, the term "region of complementarity," as defined herein, refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an RBP4 nucleotide sequence. If the region of complementarity is not perfectly complementary to the target sequence, the mismatch can be in an internal region or a terminal region of the molecule. Generally, the most tolerable mismatches are in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5' or 3' end of the iRNA. In some embodiments, a double-stranded RNA agent of the invention contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, a double-stranded RNA agent of the invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the antisense strand, e.g., the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is within, e.g., 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is within, e.g., the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0153] Thus, the RNAi agents described herein may contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can optionally be limited to be within the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand that is complementary to a region of the RBP4 gene generally does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting expression of the RBP4 gene. It is important to consider the efficacy of mismatched RNAi agents to inhibit expression of the RBP4 gene, especially when specific regions of complementarity in the RBP4 gene are known to have polymorphic sequence variation within the population.

[0154] The terms "sense strand" or "passenger strand," as used herein, refer to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0155] As used herein, "substantially all of the nucleotides are modified" means extensively but not entirely modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0156] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bounded by nucleotides 10 and 11 of the antisense strand, and this cleavage region comprises nucleotides 11, 12, and 13.

[0157] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to form a double-stranded structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions, as would be understood by one of skill in the art. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours followed by a wash (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, can be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0158] A complementary sequence within an iRNA, such as a dsRNA described herein, involves base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than five, four, three, or two, mismatched base pairs upon hybridization for a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its end use, e.g., in vitro or in vivo, for inhibiting gene expression. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0159] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed from non-naturally occurring modified nucleotides, such as, but not limited to, G:U wobble base pairs or Hoogsteen base pairs, so long as they meet the above requirements regarding their ability to hybridize.

[0160] The terms "complementary," "fully complementary," and "substantially complementary," as used herein, may be used in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA, or the antisense strand of a double-stranded RNA agent and a target sequence, as will be understood from the context of their use.

[0161] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding the RBP4 gene). For example, a polynucleotide is complementary to at least a portion of an RBP4 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the RBP4 gene.

[0162] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target RBP4 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target RBP4 sequence, and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9, or to a fragment of any one of SEQ ID NOs: 1, 3, 5, 7, or 9, over its entire length.

[0163] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target RBP4 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-3, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-3.

[0164] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to the antisense polynucleotide and thus identical to the target RBP4 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary to the equivalent region of the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, or 10, or to a fragment of any one of SEQ ID NO: 2, 4, 6, 8, or 10, over its entire length.

[0165] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target RBP4 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 2-3, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-3.

[0166] Generally, "iRNA" includes ribonucleotides that have chemical modifications. Such modifications can include all types of modifications disclosed herein or known in the art. All such modifications, when used in dsRNA molecules, are encompassed by "iRNA" for purposes of this specification and claims.

[0167] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides can be considered to constitute modified nucleotides when present within an RNAi agent.

[0168] In one embodiment of the present invention, an agent for use in the methods and compositions of the present invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotide can stoichiometrically inhibit translation by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al. (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0169] The phrase "contacting a cell with an iRNA," such as a dsRNA, as used herein encompasses contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo. Contacting can be direct or indirect. Thus, for example, an iRNA can be physically contacted with a cell by performing a method separately, or the iRNA can be placed in a situation that allows or causes it to subsequently contact a cell.

[0170] Contacting cells in vitro can be achieved, for example, by incubating cells with iRNA. Contacting cells in vivo can be achieved, for example, by injecting iRNA into or near the tissue where the cells reside, or by injecting iRNA into another region, for example, into the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue where the cells to be contacted reside. For example, iRNA can contain or be bound to a ligand, such as GalNAc, that directs iRNA to a target site, such as the liver. A combination of in vitro and in vivo contacting methods is also possible. For example, cells can be contacted with iRNA in vitro and then transplanted into a subject.

[0171] In certain embodiments, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into a cell" by promoting or causing uptake or absorption into the cell. Absorption or uptake of the iRNA can occur through spontaneous diffusion or active intracellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell can include methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below or known in the art.

[0172] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0173] As used herein, a "subject" refers to an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or a bird that expresses a target gene either endogenously or heterologously. In one embodiment, the subject is a human, e.g., a human being treated or evaluated for a disease or disorder that would benefit from reduced TTR and / or RBP4 expression, a human being at risk for a disease or disorder that would benefit from reduced TTR and / or RBP4 expression, a human having a disease or disorder that would benefit from reduced TTR and / or RBP4 expression, or a human being treated for a disease or disorder that would benefit from reduced RBP4 expression, as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

[0174] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result, such as reducing at least one sign or symptom of Stargardt disease or an RBP4-associated disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with unwanted TTR and / or RBP4 expression, reducing the degree of unwanted TTR and / or RBP4 activation or stabilization, or ameliorating or alleviating unwanted TTR and / or RBP4 activation or stabilization. "Treatment" can also mean increasing survival time compared to expected survival time if no treatment is administered.

[0175] The term "lower" in the context of TTR and / or RBP4 levels or disease markers or symptoms in a subject refers to a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., at the protein level or gene expression level. "Lower" in the context of TTR and / or RBP4 levels in a subject preferably refers to a decrease to a level that is accepted as being within the normal range for individuals without such disorders. In certain embodiments, "lower" refers to a decrease in the difference between the level of the marker or symptom in a subject suffering from the disease and a level that is accepted as being within the normal range for the individual. The term "reduce" can also be used in reference to normalizing a symptom or pathology of a disease, i.e., reducing the difference between levels in a subject afflicted with Stargardt disease or an RBP4-related disorder toward or relative to levels in a normal subject not afflicted with Stargardt disease or an RBP4-related disorder. As used herein, if the disease involves an elevated value for a symptom, "normal" refers to the upper limit of normal. If the disease involves a decreased value for a symptom, "normal" refers to the lower limit of normal.

[0176] As used herein, "prevention" or "preventing" when used in reference to a disease, disorder, or condition thereof, refers to a reduced likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition that may be treated or ameliorated by reduced expression of the TTR and / or RBP4 genes, e.g., Stargardt's disease or an RBP4-associated disorder, e.g., eye diseases such as the formation of toxic vitamin A metabolites in the retina and / or vision loss, diabetic retinopathy, age-related macular degeneration (AMD) such as dry AMD and wet AMD, iris coloboma, comedone acne syndrome, microphthalmia, basement membrane drusen, diabetic macular edema, or retinal vein occlusion, or metabolic disorders such as impaired glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, or symptoms of cardiovascular disease. Non-development of a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% on a clinically acceptable scale for the disease or disorder), or a delay in the onset of symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0177] As used herein, the term "Stargardt disease" refers to a genetic eye disorder that causes retinal degeneration and vision loss. Stargardt disease is a form of macular degeneration, also known as juvenile macular degeneration or "Stargardt macular degeneration." Stargardt disease is the most common form of hereditary macular degeneration, affecting approximately 30,000 people in the United States. The progressive vision loss associated with Stargardt disease is caused by degeneration of photoreceptor cells in the central part of the retina, called the macula. The macula is responsible for sharp central vision, which is required for detailed tasks such as reading, driving, and recognizing faces. In most people with Stargardt disease, a fatty yellow pigment (lipofuscin) accumulates in cells beneath the macula. Over time, the abnormal accumulation of this substance can damage cells that are important for clear central vision. In addition to central vision loss, people with Stargardt disease have night vision problems that can make driving in low light difficult. Some affected individuals also have impaired color vision. The signs and symptoms of Stargardt disease typically appear in late childhood or early adulthood and worsen over time.

[0178] Increased synthesis and excessive accumulation of cytotoxic lipofuscin, e.g., lipid-protein-retinoid aggregates, within the RPE are responsible for the development of Stargardt disease in a mouse model (Abca4 - / - ) demonstrated that the major cytotoxic components of RPE lipofuscin are bisretinoids. Lipofuscin synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE, and the formation of a tertiary RBP4 / TTR / retinol complex in serum is required for this influx.

[0179] As used herein, the term "retinal binding protein 4-associated disorder" or "RBP4-associated disorder" refers to a disease or disorder caused by or associated with RBP4 gene expression or RBP4 protein production. The term "RBP4-associated disorder" includes diseases, disorders, or conditions that would benefit from reduced RBP4 gene expression, replication, or protein activity. In some embodiments, the RBP4-associated disorder is an ocular disease, e.g., Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, iris coloboma, comedonal acne syndrome, microphthalmia, basement membrane drusen, diabetic macular edema, or retinal vein occlusion. In some embodiments, the RBP4-associated disorder is a metabolic disorder, e.g., a glucose and lipid homeostasis disorder, e.g., insulin resistance associated with type II diabetes, or a cardiovascular disease.

[0180] As used herein, the term "age-related macular degeneration" (AMD) or "macular degeneration" refers to the progressive degeneration of the macula, the central part of the retina, in people over the age of 55. AMD accounts for 8.7% of all blindness worldwide. AMD is characterized by large drusen deposits (deposits containing lipids and proteins) under the retina. When AMD damages the macula, the central part of a person's vision can become blurred or wavy, and blind spots can develop. AMD can cause rapid or gradual loss of vision, making it very difficult to do activities that require sharp vision, such as reading, sewing, cooking, or driving. It can also make it difficult to see in dim light. There are two types of AMD: wet AMD and dry AMD.

[0181] Macular degeneration is initiated and sustained by the accumulation of toxic vitamin A derivatives in the retinal pigment epithelium (RPE). Pharmacological inhibition of vitamin A delivery or metabolism within the RPE can significantly delay and reduce vision loss in animal models of macular degeneration. An inhibitory peptide that blocks the interaction between RBP4 and its receptor, STRA6, has been shown to reduce vitamin A delivery to the RPE and could serve as the basis for the development of therapeutic agents to treat macular degeneration (Farjo, et al., 2013, ARVO Annual Meeting, 54(15), 1702).

[0182] "Wet AMD," also known as "neovascular AMD" or "wet macular degeneration," is characterized by pathological blood vessel growth from the choroid into the retina (choroidal neovascularization), driven primarily by excessive vascular endothelial growth factor (VEGF) production by the retinal pigment epithelium (RPE).

[0183] "Dry AMD," also known as "geographic atrophy" or "dry macular degeneration," is caused by RPE cell death and photoreceptor degeneration, leading to vision loss.

[0184] As used herein, the term "diabetic retinopathy" ("DR") refers to an ocular condition that can cause vision loss and blindness in people with diabetes. It is caused by damage to the blood vessels in the light-sensitive tissue at the back of the eye (retina). Initially, diabetic retinopathy may cause no symptoms or only mild vision problems. Ultimately, it can lead to blindness. Despite intensive glycemic control, 80% of patients with type 2 diabetes progress to DR within 15 years of disease onset. Furthermore, diabetic retinopathy develops in 70-100% of people with type 1 diabetes. In the early stages, patients may present with microaneurysms, hard exudates, hemorrhages, and cotton wool spots at the back of the eye. As the disease progresses, new blood vessels may grow due to ischemia, but they are fragile and can cause bleeding and ultimately destroy the retina. The management of DR revolves around panretinal photocoagulation for proliferative disease, while diabetic macular edema is treated with focal or grid laser therapy, as well as intraocular anti-VEGF agents and steroids. Current DR therapies are associated with inconvenient delivery (laser surgery, frequent intraocular injections) and unpleasant side effects (steroid-induced glaucoma and cataracts). Recent studies have shown that RBP4 plasma levels are associated with diabetic retinopathy in patients with type 2 diabetes, and patients with higher levels of RBP4 are at higher risk of developing diabetic retinopathy (Li et al., Biosci Rep. 2018 Oct 31;38(5):BSR20181100), suggesting a potential role for RBP4 in the pathogenesis of DR complications.

[0185] As used herein, the term "diabetic macular edema" ("DME") refers to a form of diabetic retinopathy (DR) in which diseased blood vessels in the retina leak fluid from the circulation into the macula, resulting in severe vision loss.

[0186] As used herein, the term "basement membrane drusen" ("BLD"), also known as "cuticular drusen" or "early adult-onset classified drusen," refers to a condition in which small drusen are randomly deposited within the macula. In later stages, these drusen become more numerous and scattered throughout the retina, which can eventually lead to severe pigment epithelial detachment of the macula and result in vision loss. Drusen deposits are often autofluorescent.

[0187] As used herein, the term "retinal vein occlusion" ("RVO") refers to a blockage of a small vein that carries blood away from the retina, which is subdivided into central RVO and branch RVO. Central RVO is caused by impaired outflow from the central retinal vein, while branch RVO occurs when a branch of the central vein becomes blocked. Blockage can cause the retina to develop ischemia, resulting in increased VEGF and inflammatory proteins, which can promote the development of macular edema, neovascularization, glaucoma, and ultimately, blindness if left untreated. While RVO blockage cannot be cured, complications can be managed with methods such as focal laser treatment for macular edema or anti-VEGF for neovascularization.

[0188] "RBP4-associated disorder" includes any ocular disease associated with the RBP4 gene or protein in the eye that would benefit from reduced RBP4 expression. Such RBP4-associated ocular diseases are characterized, for example, by the accumulation of lipofuscin pigment (Stargardt's disease), the deposition of by-products of ocular cell metabolism called drusen in the macula (AMD and BLD), or neovascularization in the choroid or retina that accumulates and leads to impaired light transmission, tissue damage, and visual impairment or loss (AMD, DR, DME, RVO).

[0189] Additional symptoms for RBP4-associated eye diseases include, for example, difficulty seeing in the center of the vision needed for reading, sewing, cooking, seeing faces, and driving, difficulty seeing in dim light, detecting small blind spots, blurred and wavy vision, decreased dark adaptation, light sensitivity, decreased color vision, or floating spots or dark lines. Further details regarding the signs and symptoms of various diseases or conditions are provided herein and are known in the art.

[0190] In addition to eye diseases, RBP4 is also involved in various human metabolic disorders, such as impaired glucose and lipid homeostasis and cardiovascular disease.For example, RBP4 has been shown to be involved in the incidence and development of insulin resistance and diabetes (Yang, Q., et al. (2005). Nature 436, 356-362); Graham TE., et al., 2006, N Engl J Med; 354: 2552-2563).In particular, it has been reported that the serum concentration of RBP4 is elevated in insulin-resistant individuals with obesity, impaired glucose tolerance, and type 2 diabetes, as well as in lean, normoglycemic subjects with a strong family history of type 2 diabetes (Yang, Q., et al. (2005). Nature 436, 356-362); Graham TE., et al., 2006, N Engl J Med; 354: 2552-256). Transgenic overexpression of RBP4 or injection of human RBP4 in normal mice has been shown to cause insulin resistance. In contrast, genetic deletion of RBP4 or reduction of circulating RBP4 levels had the opposite effect, protecting mice from developing insulin resistance (Yang, Q., et al. (2005). Nature 436, 356-362).

[0191] Serum RBP4 has also been shown to be associated with established cardiovascular disease risk factors, including metabolic syndrome, systemic / central obesity, dyslipidemia, inflammatory markers, and hypertension (Qi Q, et al., J Clin Endocrinol Metab. 2007;92:4827-4834; Ingelsson E, et al., Atherosclerosis. 2009;206:239-244). Furthermore, RBP4 has been suggested to act as an adipokine, linking obesity to insulin resistance. Positive associations were found between serum RBP4 and adipose RBP4 mRNA and intraperitoneal fat mass, whereas serum RBP4 was inversely correlated with insulin sensitivity and decreased with exercise (Kloting et al. (2007) Cell Metab. 6, 79-87; Graham et al., 2006, N Engl J Med; 354:2552-2563). Additional studies have shown that RBP4 levels not only correlate with indicators of obesity and insulin resistance, but also with inflammatory factors (Balagopal P, et al., J Clin Endocrinol Metab 2007; 92:1971-1974), suggesting that RBP4 may be involved in the inflammatory process in diabetic retinopathy.

[0192] As used herein, "metabolic disorder" refers to any disease or disorder that disrupts normal metabolism, the process of converting food into energy at the cellular level. Metabolic disorders affect the ability of cells to carry out important biochemical reactions involving the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). In some embodiments, the RBP4-associated disorder is a metabolic disorder, e.g., a disorder of glucose and lipid homeostasis, or a cardiovascular disease.

[0193] As used herein, "disorders of glucose and lipid homeostasis" refers to any disease or disorder that disrupts normal glucose and / or lipid metabolism. Examples of disorders of glucose and lipid homeostasis include type I diabetes, type II diabetes, galactosemia, hereditary fructose intolerance, fructose-1,6-diphosphatase deficiency, glycogen storage disorders, congenital glycosylation disorders, insulin resistance, insulin deficiency, hyperinsulinemia, impaired glucose tolerance (IGT), atherosclerosis, dyslipidemia, hypertriglyceridemia (drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcoholic hypertriglyceridemia, beta-blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, and glucocorticoid-induced hypertriglyceridemia). , retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, as well as familial hypertriglyceridemia, acute pancreatitis with hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), hyperlipidemia with heterogeneous LPL deficiency, and hyperlipidemia with hyperLDLemia and heterogeneous LPL deficiency, fatty liver disease, or non-alcoholic steatohepatitis (NASH).

[0194] As used herein, the term "diabetes" refers to a group of metabolic diseases characterized by high blood sugar (glucose) levels resulting from defects in insulin secretion or action, or both. There are two most common types of diabetes: type 1 diabetes and type 2 diabetes, both of which result from the body's inability to regulate insulin. Insulin is a hormone released by the pancreas in response to increased blood sugar (glucose) levels in the blood.

[0195] As used herein, the term "type 1 diabetes" refers to a chronic disease that occurs when the pancreas produces too little insulin to properly regulate blood glucose levels. Type 1 diabetes is also known as insulin-dependent diabetes mellitus, IDDM, and juvenile-onset diabetes. People with type 1 diabetes (insulin-dependent diabetes) produce little or no insulin. Approximately 6 percent of the U.S. population has some form of diabetes, but only about 10 percent of all diabetic patients have type 1 disease. Most people with type 1 diabetes develop the disease before the age of 30. Type 1 diabetes represents the result of progressive autoimmune destruction of pancreatic beta cells with subsequent insulin deficiency. More than 90% of the pancreas' insulin-producing cells (beta cells) are permanently destroyed. The resulting insulin deficiency is severe, and type 1 diabetic patients must receive regular insulin injections to survive.

[0196] In type II diabetes (also known as non-insulin-dependent diabetes mellitus, or NDDM), the pancreas continues to produce insulin, sometimes at higher-than-normal levels. However, the body develops insulin resistance and dysregulation of insulin secretion. Type II diabetes can develop in children and adolescents, but usually begins after age 30 and becomes increasingly more common with age; approximately 15% of people over the age of 70 have type II diabetes. Obesity is a risk factor for type II diabetes, and 80-90% of people with this disorder are obese. In some embodiments, diabetes includes prediabetes. Prediabetes refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or impaired fasting glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for the development of type 2 diabetes, cardiovascular disease, and death. Much focus has been placed on developing therapeutic interventions to effectively treat prediabetes and thereby prevent the onset of type 2 diabetes.

[0197] Diabetes can be diagnosed by performing a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Primary examples of these categories include autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (NDDM type 1), insulin-dependent diabetes mellitus (IDDM type 2), non-autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (NIDDM type 2), and maturity-onset diabetes of the young (MODY). A further category, often referred to as secondary, refers to diabetes resulting from some identifiable condition that causes or allows the development of a diabetic syndrome. Examples of secondary categories include diabetes caused by pancreatic disease, diabetes induced by hormonal abnormalities, drugs, or chemicals, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes (see, e.g., Harrison's (1996) 14th ed., New York, McGraw-Hill).

[0198] Cardiovascular diseases, as defined herein, are also considered "metabolic disorders." These diseases can include coronary artery disease (also called ischemic heart disease), inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, and stroke.

[0199] Weight-related disorders are also considered "metabolic disorders" as defined herein. Such disorders can include obesity, metabolic syndrome, including its independent components (e.g., central obesity, FBG / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, urinary tract infections, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintaining weight loss, or risk of weight loss after weight loss.

[0200] As used herein, the term "agents that inhibit the expression and / or activity of transthyretin (TTR)" refers to any agent that reduces the expression and / or activity of TTR. TTR is a thyroxine transport protein and a binding partner of RBP4, which is involved in the transport of retinol in the circulation. RBP4 binding to TTR reduces the glomerular filtration rate of RBP4 and retains it in the blood. Therefore, TTR binding is an important determinant of serum RBP4 levels. When retinol binds to RBP4 in hepatocytes, a retinol / RBP4 / TTR complex is formed, released into the bloodstream, and delivers retinol to tissues via binding to specific membrane receptors. Inhibiting the expression and / or activity of TTR can also reduce the level of RBP4. Therefore, agents that inhibit the expression and / or activity of TTR can also be used to inhibit the expression and / or activity of RBP4 and can be used in methods for treating or preventing RBP4-related disorders, as described herein.

[0201] In some embodiments, agents that inhibit TTR expression and / or activity inhibit or reduce binding of TTR to RBP4, inhibit or reduce binding of retinol to TTR / RBP4, inhibit or reduce formation of a retinol / RBP4 / TTR complex, or inhibit or reduce transport or delivery of retinol to a target tissue. Exemplary agents that inhibit TTR expression and / or activity can include, but are not limited to, TTR small molecule inhibitors, nucleic acid agents that target TTR, such as siRNAs, or antisense oligonucleotides, or gene therapies that target TTR, or anti-TTR antibodies.

[0202] In some embodiments, the nucleic acid agent targeting TTR is a TTR-targeting siRNA, or antisense oligonucleotide, or gene therapy.

[0203] In one embodiment, the nucleic acid agent targeting TTR is butrisilane.

[0204] In one embodiment, the nucleic acid agent is a dsRNA agent, or a salt thereof, comprising a sense strand comprising the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO:20), and an antisense strand comprising the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO:21), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: [ka] In the formula, X is O.

[0205] In one embodiment, the TTR-targeting nucleic acid is revusiran.

[0206] In one embodiment, the nucleic acid agent is a dsRNA agent comprising a sense strand comprising the nucleotide sequence 5'-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAf-3' (SEQ ID NO:22), and an antisense strand comprising the nucleotide sequence 5'-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3' (SEQ ID NO:23), or a salt thereof, wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U; s is a phosphorothioate linkage; and the ligand is conjugated to the 3' end of the sense strand as shown in the following schematic diagram: [ka] In the formula, X is O.

[0207] In one embodiment, the nucleic acid agent targeting TTR is patisiran.

[0208] In one embodiment, the nucleic acid agent is a dsRNA agent comprising a sense strand comprising the nucleotide sequence 5'-GuAAccAAGAGuAuuccAudTdT-3' (SEQ ID NO: 24) and an antisense strand comprising the nucleotide sequence 5'-AUGGAAuACUCUUGGUuACdTdT-3' (SEQ ID NO: 25), or a salt thereof, where A is adenosine, C is cytidine, G is guanosine, U is uridine, a is 2'-O-methyladenosine, c is 2'-O-methylcytidine, g is 2'-O-methylguanosine, u is 2'-O-methyluridine, and dT is 2'-deoxythymidine.

[0209] Suitable dsRNA agents suitable for use in the claimed methods are described in the disclosures of PCT Publication Nos. 2013 / 075035, 2017 / 023660, and 2010 / 048228, the entire contents of which are incorporated herein by reference.

[0210] In one embodiment, the nucleic acid agent targeting TTR is inotersen.

[0211] In one embodiment, the nucleic acid agent is a single-stranded modified oligonucleotide of 20 linked nucleosides having a nucleobase sequence of 5'-TCTTGGTTACATGAAATCCC-3' (SEQ ID NO: 26), wherein the modified oligonucleotide comprises a gap segment of 10 linked deoxynucleosides, a 5' wing segment of 5 linked nucleosides, and a 3' wing segment of 5 linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage, and each cytosine in the modified oligonucleotide is a 5-methylcytosine.

[0212] In yet another embodiment, the agent that inhibits the expression and / or activity of TTR is a stabilizer of the quaternary structure of the transthyretin protein, e.g., tafamidis (see, e.g., U.S. Patent Nos. 8,653,119, 8,168,663, 7,214,696, and 7,214,695, the entire contents of each of which are incorporated herein by reference).

[0213] A "therapeutically effective amount," as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject with Stargardt disease or an RBP4-associated disorder, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining the existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the subject to be treated.

[0214] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with Stargardt disease or an RBP4-associated disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the progression of the disease or reducing the severity of any future disease. A "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if applicable, and other personal characteristics of the patient to be treated.

[0215] A "therapeutically effective amount" or a "prophylactically effective amount" also includes that amount of an RNAi agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNAs used in the methods of the invention can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0216] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0217] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transfer of a subject compound from one organ or body part to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0218] The term "sample," as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or bodily fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the entire liver or a specific segment of the liver, or a specific type of cell within the liver, such as a hepatocyte). In certain embodiments, samples can be derived from the retina or a portion of the retina (e.g., the retinal pigment epithelium and / or the ciliary epithelium). In some embodiments, a "sample derived from a subject" refers to retinal tissue obtained from a subject. In some embodiments, a "sample derived from a subject" refers to urine obtained from a subject. A "sample derived from a subject" can also refer to blood from a subject or serum or plasma derived from blood.

[0219] II. iRNAs of the Invention In certain aspects, the present invention provides iRNAs that inhibit expression of the RBP4 gene. In certain embodiments, the iRNAs comprise double-stranded ribonucleic acid (dsRNA) molecules for inhibiting expression of the RBP4 gene in cells, e.g., in a subject, e.g., in a mammal, e.g., in a cell with an RBP4-associated disorder, e.g., an ocular disease, e.g., Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, or in a human susceptible to developing a metabolic disorder, e.g., a disorder of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, or cardiovascular disease. The dsRNAi agent comprises an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed upon expression of the RBP4 gene. The region of complementarity is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0220] When contacted with cells expressing the RBP4 gene, the iRNA inhibits expression of the RBP4 gene (e.g., human, primate, non-primate, or rat RBP4 gene) by at least about 50%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, for example, by immunofluorescence analysis, e.g., using Western blot or flow cytometry techniques. In certain embodiments, inhibition of expression is determined by the qPCR method provided in the Examples herein, using siRNA at a concentration of, for example, 10 nM, in an appropriate biological cell line provided therein. In certain embodiments, inhibition of expression in vivo is determined by knocking down the human gene in rodents expressing the human gene, e.g., mice expressing a human target gene or AAV-infected mice, when administered, e.g., as a single dose, at a minimum of 3 mg / kg of RNA expression.

[0221] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which dsRNA will be used. One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary, and generally completely complementary, to the target sequence. The target sequence can be obtained from the sequence of mRNA formed during the expression of the RBP4 gene. The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequences of dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, such that they are opposite each other on separate oligonucleotides.

[0222] Generally, the duplex structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 19-6 The length is 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also contemplated as part of this disclosure.

[0223] Similarly, the region of complementarity to the target sequence may be 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 , 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths that lie between the ranges and lengths listed above are also intended to be part of this disclosure.

[0224] In some embodiments, the duplex structure is 19-30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19-30 nucleotides in length.

[0225] In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides in length can function as substrates for Dicer. As those skilled in the art will recognize, the region of an RNA targeted for cleavage is almost always a portion of a longer RNA molecule, often an mRNA molecule. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0226] Those skilled in the art will understand that a double-stranded region is the primary functional portion of a dsRNA, e.g., a double-stranded region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. That is, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, so long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, one of skill in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting RBP4 gene expression is not generated in the target cell by cleavage of a larger dsRNA.

[0227] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1 to 4, 2 to 4, 1 to 3, 2 to 3, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Moreover, the overhanging nucleotide may be present on the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the dsRNA.

[0228] dsRNA can be synthesized by standard methods known in the art.Double-stranded RNAi compounds of the present invention can be prepared using a two-step method.First, each strand of double-stranded RNA molecules is prepared separately.These component strands are then annealed.The individual strands of siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare oligonucleotide strands containing unnatural nucleotides or modified nucleotides.Similarly, single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0229] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences: a sense sequence and an antisense sequence. The sense strand is selected from the sequences provided in any one of Tables 2-3, and the corresponding antisense strand of the sense strand is selected from the sequences provided in any one of Tables 2-3. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of mRNA produced upon expression of the RBP4 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide being listed in any one of Tables 2-3 as the sense strand and the second oligonucleotide being listed in any one of Tables 2-3 as the corresponding antisense strand of the sense strand.

[0230] In certain embodiments, the substantially complementary sequences of the dsRNA are contained on each oligonucleotide. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0231] For example, although the sequences in Table 3 are not described as modified or conjugated, the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, can comprise any one of the sequences set forth in any one of Tables 2-3, unmodified, unconjugated, or modified or conjugated differently than those set forth therein. In other words, the invention encompasses the dsRNA of Tables 2-3, unmodified, unconjugated, modified, or conjugated, as described herein.

[0232] Those skilled in the art are well aware that dsRNAs having duplex structures of about 20 to 23 base pairs, for example, 21 base pairs, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have discovered that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of the oligonucleotide sequences provided in any one of Tables 2-3, the dsRNAs described herein can comprise at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes having any one of the sequences in any one of Tables 2-3, minus only a few nucleotides on one or both ends, can be similarly effective compared to the above-mentioned dsRNAs. Thus, dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides derived from any one of the sequences in any one of Tables 2-3 and that differ from dsRNAs containing the entire sequence in their ability to inhibit expression of the RBP4 gene by no more than about 5, 10, 15, 20, 25, or 30% inhibition are contemplated within the scope of the present invention.

[0233] Additionally, the RNAs provided in Tables 2-3 identify sites within the RBP4 transcript that are susceptible to RISC-mediated cleavage. Thus, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within the specific site. Such iRNAs will generally comprise at least about 19 contiguous nucleotides from any one of the sequences provided in any one of Tables 2-3 linked to additional nucleotide sequences taken from regions adjacent to the selected sequence within the RBP4 gene.

[0234] III. Modified iRNAs of the Invention In certain embodiments, the RNA, e.g., dsRNA, of an iRNA of the invention is unmodified, e.g., does not contain chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA, e.g., dsRNA, of an iRNA of the invention is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA, or substantially all of the nucleotides of an iRNA, are modified, i.e., no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 unmodified nucleotide is present in any strand of the iRNA.

[0235] Nucleic acids featured in the present invention can be synthesized or modified by methods such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference in its entirety. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted ligation) or 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.), base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base pair with partners in an extended repertoire, base removal (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or backbone modifications, including modifications or substitutions of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or containing non-natural internucleoside linkages. Among the RNAs with modified backbones, those that do not have a phosphorus atom in their backbones are included.For the purposes of this specification, as sometimes referred to in the art, the modified RNA that does not have a phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified iRNA will have a phosphorus atom in its internucleoside backbone.

[0236] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agents of the present invention are in the free acid form. In other embodiments of the present invention, the dsRNA agents of the present invention are in the salt form. In one embodiment, the dsRNA agents of the present invention are in the sodium salt form. In certain embodiments, when a dsRNA agent of the invention is in sodium salt form, sodium ions are present in the agent as counterions to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent. An agent in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterions includes 5, 4, 3, 2, or 1 or fewer phosphodiester and / or phosphorothioate linkages that do not have sodium counterions. In some embodiments, when a dsRNA agent of the invention is in sodium salt form, sodium ions are present in the agent as counterions to all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0237] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, and 5,286,717. , No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 ,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. No. 5,587,361, No. 5,625,050, No. 6,028,188, No. 6,124,445, No. 6,160,109, No. 6,169,170, No. 6,172,20 No. 9, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,5 Nos. 34,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0238] Modified RNA backbones that do not contain a phosphorus atom in the backbone have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0239] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0240] RNA mimics suitable for use in the iRNAs provided herein are contemplated in which both the sugar and internucleoside linkage of the nucleotide units, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimic known to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0241] Some embodiments featured herein include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as the methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of the above-referenced U.S. Patent No. 5,489,677, and the amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Patent No. 5,034,506. The natural phosphodiester backbone can be represented as OP(O)(OH)-OCH2-.

[0242] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2' position: OH; F, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, interfering substances, groups for improving the pharmacokinetic properties of iRNA or groups for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers within these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0243] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNA, and the 5' position of 5' terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include U.S. Patent 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, Nos. 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, portions of which are commonly owned with this application, the entire contents of each of the foregoing being incorporated herein by reference.

[0244] iRNAs may also contain modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and the like. Examples include cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are a further exemplary base substitution, particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0245] Representative United States patents that teach the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-referenced U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469 ... Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0246] In some embodiments, RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a ring formed by a bridge between two carbon atoms, whether adjacent or non-adjacent. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a ring formed by bridging two carbon atoms of the sugar ring, whether adjacent or non-adjacent, thereby forming a bicyclic ring system. In certain embodiments, a bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally through a 2'-acyclic oxygen atom. Thus, in some embodiments, an agent of the present invention can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo conformation. The addition of a locking nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides containing a 4' to 2' bridge.

[0247] A locked nucleoside can be represented by this structure (stereochemistry omitted): [ka] wherein B is a nucleobase or modified nucleobase, and L is a linking group connecting the 2'-carbon and 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt"), and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs thereof, e.g., U.S. Pat. No. 7,399,845). No. 8,278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a nitrogen protecting group (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH2-C(=CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0248] Additional representative U.S. patents and publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, Nos. 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618 and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0249] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0250] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge (L in the preceding structure). In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0251] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0252] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383 and PCT Publication No. 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0253] In some embodiments, the iRNA of the invention includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are non-locked acyclic nucleic acids in which any of the sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also include monomers in which the C1'-C4' linkage has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' linkage of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0254] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0255] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in PCT Publication No. 2011 / 005861.

[0256] Other modifications of nucleotides of the iRNAs of the invention include 5' phosphates or 5' phosphate mimics, such as 5' terminal phosphates or phosphate mimics on the antisense strand of the iRNA. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0257] A. Modified iRNAs Containing Motifs of the Invention In certain aspects of the present invention, the double-stranded RNA agent of the present invention includes an agent with chemical modifications, such as those disclosed in WO2013 / 075035, the entire contents of which are incorporated herein by reference.As shown herein and in WO2013 / 075035, one or more motifs of three identical modifications on three consecutive nucleotides can be introduced into the sense strand or antisense strand of dsRNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of dsRNAi agent can be otherwise completely modified.The introduction of these motifs interrupts the modification pattern of the sense strand or antisense strand, if present.This dsRNAi agent can optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand.

[0258] More specifically, gene silencing activity of a dsRNAi agent was observed when the sense and antisense strands of the double-stranded RNA agent were fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0259] Thus, the present invention provides double-stranded RNA agents capable of inhibiting expression of a target gene (i.e., the RBP4 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0260] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as a "dsRNAi agent." The double-stranded region of a dsRNAi agent can be, for example, a double-stranded region that can be 27-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the double-stranded region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0261] In certain embodiments, a dsRNAi agent can contain one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can independently be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. In certain embodiments, the overhang region can include an extended overhang region, as described above. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be a different sequence. The first and second strands can also be joined by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0262] In certain embodiments, the nucleotides in the overhang region of a dsRNAi agent can each independently be modified or unmodified nucleotides, including, but not limited to, a 2'-sugar modification, including, for example, 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0263] For example, TT can be an overhang sequence for either end on either strand, which can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0264] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of a dsRNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate between them, and the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In some embodiments, the 3'-overhang is present in the antisense strand. In some embodiments, the 3'-overhang is present in the sense strand.

[0265] dsRNAi agent can only contain a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, single-stranded overhang can be located at the 3' end of sense strand or at the 3' end of antisense strand.RNAi can also have a blunt end, which is located at the 5' end of antisense strand (i.e., the 3' end of sense strand), or vice versa.Generally, the antisense strand of dsRNAi agent has a nucleotide overhang at the 3' end, and its 5' end is blunt.Without wishing to be bound by theory, the blunt end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are asymmetric, which is favorable for the insertion of guide strand into RISC process.

[0266] In certain embodiments, the dsRNAi agent is 19 nucleotides in length and double-blunt ended, and the sense strand contains at least one motif of three 2'-F modifications on 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 on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0267] In other embodiments, the dsRNAi agent is 20 nucleotides in length and double-blunt ended, and the sense strand contains at least one motif of three 2'-F modifications on 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 on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0268] In yet other embodiments, the dsRNAi agent is 21 nucleotides in length and double-blunt ended, and the sense strand comprises at least one motif of three 2'-F modifications on 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 on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0269] In certain embodiments, the dsRNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on 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 on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt and the other end comprises a two-nucleotide overhang. In one embodiment, the two-nucleotide overhang is at the 3' end of the antisense strand.

[0270] When a two-nucleotide overhang is at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In certain embodiments, all nucleotides in the sense strand and antisense strand of the dsRNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In certain embodiments, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. Optionally, the dsRNAi agent further comprises a ligand (e.g., GalNAc3).

[0271] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and, starting from the 5'-most nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1 to 23 of the first strand; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-most nucleotide, comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand, forming a duplex; at least the 3'-most nucleotide of the antisense strand is unpaired with the sense strand, and up to six consecutive 3'-most nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5'-end of the antisense strand is 10 to 30 nucleotides that are not paired with the sense strand. The double-stranded nucleic acid comprises at least 10 ribonucleotides of the 5'- and 3'-terminal nucleotides of the sense strand, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, such that when the double-stranded nucleic acid is introduced into a mammalian cell, target gene expression is reduced, and the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0272] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, the dsRNAi agent comprising a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand form blunt ends, and the second strand is 1 to 4 nucleotides longer at its 3' end than the first strand, the double-stranded region is at least 25 nucleotides in length, the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the second strand, the RNAi agent reduces target gene expression when introduced into a mammalian cell, and Dicer cleavage of the dsRNAi agent results in an siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNAi agent further comprises a ligand.

[0273] In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at the cleavage site in the sense strand.

[0274] In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which occurs at or near the cleavage site in the antisense strand.

[0275] For dsRNAi agents having a double-stranded region 19-23 nucleotides in length, the cleavage sites in the antisense strand are typically located at approximately positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, where the numbers start from the first nucleotide from the 5' end of the antisense strand, or the numbers start from the first paired nucleotide in the double-stranded 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 double-stranded region of the dsRNAi agent from the 5' end.

[0276] The sense strand of dsRNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the breakpoint of strand, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the breakpoint of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairing.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0277] In some embodiments, the sense strand of a dsRNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand, and the other motif may be a wing modification. As used herein, the term "wing modification" refers to a motif occurring in another part of the strand separated from the motif at or near the cleavage site of the same strand. The wing modification may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical nature of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemical nature may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0278] Similar to the sense strand, the antisense strand of dsRNAi agent can contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand breakage.This antisense strand can also contain one or more wing modifications in the same sequence as the wing modifications that can be present on the sense strand.

[0279] In some embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0280] In other embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two paired nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0281] When the sense and antisense strands of a dsRNAi agent each include at least one wing modification, the wing modifications can be at the same end of the double-stranded region and have an overlap of 1, 2, or 3 nucleotides.

[0282] When the sense or antisense strand of a dsRNAi agent each contains at least two wing modifications, the sense and antisense strands can be aligned such that two modifications from each single strand are at one end of a double-stranded region with an overlap of 1, 2, or 3 nucleotides, two modifications from each single strand are at the other end of a double-stranded region with an overlap of 1, 2, or 3 nucleotides, and two modifications from each single strand are on either side of a lead motif within the double-stranded region with an overlap of 1, 2, or 3 nucleotides.

[0283] In some embodiments, any nucleotide in the sense strand and antisense strand of the dsRNAi agent, including the nucleotide that is part of the motif, can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include: changing one or both of the non-binding phosphate oxygens or one or more of the binding phosphate oxygens; changing the ribose sugar component, for example, the 2'-hydroxyl on the ribose sugar; extensively replacing the phosphate moiety with a "dephosphorylation" linker; modifying or replacing naturally occurring bases; and replacing or modifying the ribose phosphate backbone.

[0284] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties, or non-linked Os in phosphate moieties. In some cases, modifications will occur at all target positions in a nucleic acid, but in many cases, this is not the case. For example, modifications can occur only at the 3'- or 5'-terminal positions, or only in terminal regions, such as at the terminal nucleotide of the chain or in the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only within the double-stranded region of RNA, or only within the single-stranded region of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide of the chain or in the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in both double-stranded and single-stranded regions, especially at the ends. The 5' end or ends can be phosphorylated.

[0285] For example, to enhance stability, it may be possible to include specific bases in the overhang, or to include modified nucleotides or nucleotide substitutes in a single-stranded overhang, e.g., in the 5' or 3' overhang, or in both overhangs. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or a portion of the bases in the 3' or 5' overhang can be modified, e.g., with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar, with modifications known in the art, e.g., the use of modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, in place of the ribosugar of the nucleobase, and modifications at the phosphate group, e.g., phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0286] In some embodiments, each residue in the sense strand and the antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. A strand may contain more than one modification. In one embodiment, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0287] At least two different modifications are typically present on the sense and antisense strands, which may be 2'-O-methyl or 2'-fluoro modifications.

[0288] In certain embodiments, N a or N b includes an alternating pattern of modifications. The term "alternating motif," as used herein, refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a single strand. The alternating nucleotides can refer to one every other nucleotide, or one every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0289] The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications of the alternating motif, such as "ABABAB...," "ACACAC...," "BDBDBD...," or "CDCDCD...."

[0290] In some embodiments, the dsRNAi agent of the present invention comprises a modification pattern for the alternating motif on the sense strand that is shifted relative to the modification pattern for the alternating motif on the antisense strand. The shift can be such that the modified group of the nucleotide of the sense strand corresponds to the differently modified group of the nucleotide of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from 5' to 3' of the strand in the double-stranded region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from 5' to 3' of the strand in the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0291] In some embodiments, dsRNAi agent comprises the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification on sense strand, and first has a relative shift with respect to the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification on antisense strand, that is, comprises 2'-O-methyl modified nucleotide on base pair of sense strand and 2'-F modified nucleotide on antisense strand, and vice versa.Position 1 of sense strand can start with 2'-F modification, and position 1 of antisense strand can start with 2'-O-methyl modification.

[0292] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand or antisense strand interrupts the original modification pattern present in the sense strand or antisense strand.The interruption of the modification pattern of the sense strand or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand or antisense strand can enhance the gene silencing activity against the target gene.

[0293] In some embodiments, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different modifications from the modification of the motif. For example, the portion of the sequence containing the motif may be described as "...N a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications on three consecutive nucleotides, and "N a " and "N b " represents a modification to the nucleotide adjacent to the motif "YYY" that is different from the modification of Y, and N a and N b may be the same or different modifications. Alternatively, when wing modifications are present, N a or N b may or may not be present.

[0294] The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur at any nucleotide in the sense strand, the antisense strand, or both strands at any position in the strand. For example, the internucleotide linkage modification may occur at every nucleotide on the sense strand or the antisense strand, each internucleotide linkage modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0295] In some embodiments, the dsRNAi agent comprises 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 the two nucleotides. The internucleotide bond modification can also be made to link the overhang nucleotide to the terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked by a phosphorothioate or methylphosphonate internucleotide bond, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides can be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 5' end of the antisense strand.

[0296] In some embodiments, the 2-nucleotide overhang is at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. Optionally, the dsRNAi agent can further have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0297] In one embodiment, the dsRNAi agent contains mismatches or combinations thereof within the double strand with the target. Mismatches can occur within the overhang region or within the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs based on individual base pairs, but adjacent bases or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C, G:U is preferred to G:C, and I:C is preferred to G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are preferred to standard pairings (A:T, A:U, G:C), and pairings involving universal bases are preferred to standard pairings.

[0298] In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the double-stranded region from the 5' end of the antisense strand independently selected from the following group: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, that promote dissociation of the antisense strand at the 5' end of the duplex.

[0299] In certain embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0300] In other embodiments, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT) or the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT), for example, there is a short sequence of deoxythymidine nucleotides, e.g., two dT nucleotides on the 3' end of the sense strand, the antisense strand, or both strands.

[0301] In certain embodiments, the sense strand sequence has 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 each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide, wherein Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In one embodiment, YYY are all 2'-F modified nucleotides.

[0302] In some embodiments, N a or N b includes alternating pattern modifications.

[0303] In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, if the dsRNAi agent has a double-stranded region that is 17 to 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), where the numbers start from the first nucleotide from the 5' end, or optionally, the numbers start from the first paired nucleotide in the double-stranded region from the 5' end.

[0304] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib), 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic), or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0305] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0306] When the sense strand is represented by formula (Ic), N brepresents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0307] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0308] Each of X, Y and Z may be the same or different.

[0309] In other embodiments, i is 0 and j is 0, and the sense strand has the formula 5'n p -N a -YYY- N a -n q 3'(Ia).

[0310] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0311] In one embodiment, the antisense strand sequence of the RNAi is represented by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II), During the ceremony, k and l 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 ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide, In the formula, N b ' and Y' do not have the same modification, and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0312] In some embodiments, N a ' or N b ' includes alternating pattern modifications.

[0313] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand.For example, when the dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, where the numbers start from the first nucleotide from the 5' end, or optionally, the numbers start from the first paired nucleotide in the double-stranded region from the 5' end.In one embodiment, the Y'Y'Y' motif occurs at positions 11, 12, and 13.

[0314] In certain embodiments, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0315] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0316] Thus, the antisense strand has the following formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb), 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc), or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3'(IId).

[0317] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0318] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0319] When the antisense strand is represented by formula (IId), each N bEach N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a In one embodiment, N' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6.

[0320] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula 5'n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3'(Ia).

[0321] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X', Y' and Z' may be the same as or different from one another.

[0322] Each nucleotide in the sense strand and the antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense strand and the antisense strand can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0323] In some embodiments, the sense strand of a dsRNAi agent can contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or optionally the numbers starting from the first paired nucleotide in the double-stranded region from the 5' end, and Y represents a 2'-F modification. The sense strand can further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0324] In some embodiments, the antisense strand can contain a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, where the numbering starts from the first nucleotide from the 5' end, or optionally the numbering starts from the first paired nucleotide in the double-stranded region from the 5' end, and Y' represents a 2'-O-methyl modification. The antisense strand can further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0325] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0326] Thus, the dsRNAi agents used in the methods of the invention can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the iRNA duplex can have the following formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) During the ceremony, i, j, k, and l are each independently 0 or 1; p, p', q, and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; In the formula, each n p ',n p , n q ' and n q each of which may or may not be present independently represents an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0327] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0328] Exemplary combinations of sense and antisense strands that form iRNA duplexes include the following formulas: 5'n p - N a -YYY -N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YY YN b -ZZ ZN a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XX XN b -YY YN a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5'n p -N a -XX XN b -YY YN b -ZZ ZN a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId).

[0329] When the dsRNAi agent has formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0330] When the dsRNAi agent has formula (IIIb), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0331] When the dsRNAi agent has formula (IIIc), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0332] When the dsRNAi agent has formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b ’ each independently comprises an alternating pattern of modifications.

[0333] Each of X, Y, and Z in formulae (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from one another.

[0334] When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides, alternatively, at least two Y nucleotides are base-paired with the corresponding Y' nucleotide, or all three Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0335] When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides. Alternatively, at least two Z nucleotides can be base-paired with a corresponding Z' nucleotide, or all three Z nucleotides can be base-paired with a corresponding Z' nucleotide.

[0336] When the dsRNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, alternatively, at least two of the X nucleotides can be base-paired with the corresponding X' nucleotide, or all three of the X nucleotides can be base-paired with the corresponding X' nucleotide.

[0337] In certain embodiments, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, or the modification on a Z nucleotide is different from the modification on a Z' nucleotide, or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0338] In certain embodiments, when the dsRNAi agent has formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, and p '>0 and at least one n p In yet another embodiment, when the RNAi agent has formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate bond. a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker (described below). a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0339] In some embodiments, when the dsRNAi agent has Formula (IIIa), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0340] In some embodiments, the dsRNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), and the duplexes are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes, or each duplex may target the same gene at two different target sites.

[0341] In some embodiments, the dsRNAi agent is a multimer containing 3, 4, 5, 6, or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), and the duplexes are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes, or each duplex may target the same gene at two different target sites.

[0342] In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5' end and at one or both of the 3' ends, and are optionally conjugated to a ligand. The agents can each target the same gene or two different genes, or the agents can each target the same gene at two different target sites.

[0343] In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modifications, for example, 10 or fewer nucleotides with 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2'-fluoro modifications. In certain embodiments, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and 6 nucleotides with 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and 2 nucleotides with 2'-fluoro modifications in the antisense strand.

[0344] In other embodiments, RNAi agents of the present invention may contain very few nucleotides containing 2'-fluoro modifications, for example, two or fewer nucleotides containing 2'-fluoro modifications. For example, an RNAi agent may contain two, one, or zero nucleotides with 2'-fluoro modifications. In certain embodiments, an RNAi agent may contain two nucleotides with 2'-fluoro modifications, for example, zero nucleotides with 2'-fluoro modifications in the sense strand and two nucleotides with 2'-fluoro modifications in the antisense strand.

[0345] Various publications describe multimeric iRNAs that can be used in the methods of the invention, including WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0346] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In exemplary embodiments, a 5'-vinyl phosphonate modified nucleotide of the present disclosure has the following structure: [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the C5' carbon and R 5’ and the double bond between B is a nucleobase or a modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0347] The vinyl phosphonates of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonates of the present disclosure are attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0348] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include those described above, where R5' is =C(H)-O-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., E configuration).

[0349] As described in more detail below, iRNAs containing one or more carbohydrate moieties conjugated to the iRNA can optimize one or more properties of the iRNA. Often, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbon-cyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0350] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," e.g., 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 to a bond that is available and suitable for incorporating the carrier into the backbone of a ribonucleic acid, e.g., a phosphate, or a modified phosphate, e.g., sulfur-containing. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (other than the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers will often contain functional groups, such as amino groups, or generally provide bonds suitable for the incorporation or tethering of another chemical entity, such as a ligand, to the constituent ring.

[0351] The iRNA can be conjugated to the ligand via a carrier, which can be a cyclic or acyclic group. In one embodiment, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a serinol skeleton or a diethanolamine skeleton.

[0352] i. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermostabilizing modifications into the seed region of antisense strand.As used herein, "seed region" refers to the 2-9 positions of the 5' end of the referenced strand.For example, thermostabilizing modifications can be incorporated into the seed region of antisense strand to reduce or inhibit off-target gene silencing.

[0353] The term "thermally destabilizing modification" refers to the melting temperature (T m ) lower than the overall melting temperature (T m For example, thermally destabilizing modifications include modifications that result in dsRNAs with a T m can be decreased by 1-4° C., e.g., 1, 2, 3, or 4 degrees Celsius. Also, the term "thermally destabilized nucleotide" refers to a nucleotide that contains one or more thermally destabilizing modifications.

[0354] It has been discovered that dsRNAs having an antisense strand containing at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5, or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the one or more thermally destabilizing modifications of the duplex are located at positions 2-9, e.g., positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at positions 6, 7, or 8 from the 5' end of the antisense strand. In even some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. In some embodiments, the thermally destabilizing modification of the duplex is located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0355] An iRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. An RNAi agent has the following formula (L): [ka] (L), which can be expressed as:

[0356] In Formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In 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, 2'-O-CHC(O)N(Me)H) modification.

[0357] C1 is a thermolabile nucleotide located 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 the nucleotide at positions 2-8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 from the 5' end of the sense strand. The C1 nucleotide carries a thermolabile modification that may include an abasic modification, a mismatch with the opposing nucleotide in 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 is located at: i) a mismatch with the opposing nucleotide in 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, R 1 and R 2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar modification. In one embodiment, the thermolabile modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T, and optionally at least one nucleobase in the mismatch pair is a 2'-deoxynucleobase. In one example, the thermolabile modification in C1 is GNA or [ka] is.

[0358] 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 the steric bulk of the 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Methods for determining the steric effect of a nucleotide modification are known to those skilled in the art. The modification can be at the 2'-position of the ribose sugar of the nucleotide, or can be a non-ribose nucleotide, an acyclic nucleotide, or a modification to the backbone of the nucleotide that is similar or equivalent to the 2'-position of the ribose sugar and provides the nucleotide with steric bulk equal to or less than the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In 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.

[0359] n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length.

[0360] n 5 , q 3 , and q 7 are independently 1 to 6 nucleotides in length.

[0361] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length, or n 4 is 0 nucleotides in length.

[0362] q 5 are independently 0 to 10 nucleotides in length.

[0363] n 2 , and q 4 are independently 0 to 3 nucleotides in length.

[0364] Or, n 4 is 0 to 3 nucleotides in length.

[0365] 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, with 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).

[0366] In one embodiment, n 4 , q 2 , and q 6 are each 1.

[0367] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each 1.

[0368] In one embodiment, C1 is selected from the group consisting of a sense strand having a length of 19 to 22 nucleotides and a 4 When C1 is 1, it is at positions 14 to 17 of the 5' end of the sense strand. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0369] In one embodiment, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand, and 6 is equal to 1.

[0370] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. 2 is equal to 1.

[0371] In an exemplary embodiment, T3' starts at position 2 from the 5' end of the antisense strand, and T1' starts at position 14 from the 5' end of the antisense strand. In one example, T3' starts at position 2 from the 5' end of the antisense strand, and q 6 is equal to 1, and T1' starts at position 14 from the 5' end of the antisense strand, and q 2 is equal to 1.

[0372] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0373] In one embodiment, T1' is at position 14 from the 5' end of the antisense strand. 2 is equal to 1 and modifications at the 2' position or at non-ribose, acyclic or backbone positions are less sterically bulky than 2'-OMe ribose.

[0374] In one embodiment, T3' is at position 2 from the 5' end of the antisense strand. In one example, T3' is at position 2 from the 5' end of the antisense strand, and 6 is equal to 1, and non-ribose acyclic or backbone modifications at the 2' position are less or equally sterically bulky than 2'-OMe ribose.

[0375] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is located at the cleavage site of the sense strand, where the sense strand is 19-22 nucleotides in length and n 2 is 1, it is at position 11 from the 5' end of the sense strand. In an exemplary embodiment, T1 is at position 11 from the 5' end of the sense strand when the sense strand is 19-22 nucleotides in length and n 2When is 1, it is at the cleavage site of the sense strand, which is at position 11 from the 5' end of the sense strand.

[0376] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is at positions 6-10 from the 5' end of the antisense strand, and 4 is 1.

[0377] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., the sense strand is 19-22 nucleotides in length and 2 When T1' is 1, it is located at the 11th position from the 5' end of the sense strand, and T1' is located at the 14th position from the 5' end of the antisense strand. 2 is equal to 1, and the modification to T1' is at the 2' position of the ribose sugar or at a non-ribose acyclic or intra-backbone position that is less sterically bulky than 2'-OMe ribose, T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1, and T3' is at the second position from the 5' end of the antisense strand, and q 6 is equal to 1 and the modification to T3' is at the 2' position or at a non-ribose acyclic or intra-backbone position that is less sterically bulky than 2'-OMe ribose.

[0378] In one embodiment, T2' starts at position 8 from the 5' end of the antisense strand. 4 is 2.

[0379] In one embodiment, T2' begins at position 9 from the 5' end of the antisense strand. 4 is 1.

[0380] 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 3is 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0381] 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 q 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

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

[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, 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0384] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 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.

[0385] 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[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, 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.

[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, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[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 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 accompanied by at least two additional TTs at the 3' end of the antisense strand.

[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 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 q4 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), and with two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[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 7 is 1.

[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 q1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, with 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.

[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 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.

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

[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 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.

[0395] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1, with 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).

[0396] The RNAi agent can include a phosphorus-containing group at the 5'-end of 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] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ), or a mixture thereof.

[0397] In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0398] In one embodiment, the RNAi agent comprises a 5'-P. In one embodiment, the RNAi agent comprises a 5'-P in the antisense strand.

[0399] In one embodiment, the RNAi agent comprises a 5'-PS. In one embodiment, the RNAi agent comprises a 5'-PS in the antisense strand.

[0400] In one embodiment, the RNAi agent comprises a 5'-VP. In one embodiment, the RNAi agent comprises a 5'-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5'-Z-VP in the antisense strand.

[0401] In one embodiment, the RNAi agent comprises a 5'-PS2. In one embodiment, the RNAi agent comprises a 5'-PS2 in the antisense strand.

[0402] In one embodiment, the RNAi agent comprises a 5'-PS2. In one embodiment, the RNAi agent comprises a 5'-deoxy-5'-C-malonyl in the antisense strand.

[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 q2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0405] 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 3is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[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, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-PS2.

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

[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, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[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 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'-OMe, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

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

[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, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2.

[0412] 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 n3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0413] 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 RNAi agent also contains a 5'-P.

[0414] 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'-PS.

[0415] 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 RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0416] 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 RNAi agent also includes a 5'-PS2.

[0417] 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0418] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-P.

[0419] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-PS.

[0420] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0421] 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 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, with 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 RNAi agent also includes a 5'-PS2.

[0422] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0423] 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 RNAi agent also contains a 5'-P.

[0424] 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. The RNAi agent also contains a 5'-PS.

[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'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[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 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNAi RNA agent also includes a 5'-PS2.

[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'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[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 q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[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'-F, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

[0430] 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'-F, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[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, 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 q7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2.

[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, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[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 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. The RNAi agent also contains a 5'-P.

[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'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

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

[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 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-PS2.

[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 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[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 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

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

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

[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 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2.

[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 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 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[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, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[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, 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0445] 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, with two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0446] In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0447] 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 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0448] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi 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' end of the sense strand.

[0449] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0450] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0451] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0452] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0453] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0454] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0455] 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 q4 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0456] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0457] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0458] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi 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' end of the sense strand.

[0459] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0460] 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 3is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0461] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0462] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0463] 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, with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi 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' end of the sense strand.

[0464] In certain embodiments, the RNAi agents of the invention include: (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2'-F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end), and (iii) phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23 (counting from the 5' end); 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.

[0465] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end), and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0466] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a deoxy-nucleotide (e.g., dT) at position 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2'-F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0467] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7, 9, 11, 13, and 15, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2'-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0468] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 9, 12 to 21, and 2'-F modifications at positions 10 and 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0469] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0470] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 to 21, and 2'-F modifications at positions 3, 5, 7, 9 to 11, 13, 16, and 18, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, 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 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi 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.

[0471] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0472] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0473] In another particular embodiment, the RNAi agent of the invention is (a) a sense strand having: (i) 19 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2'-F modifications at positions 5, and 7 to 9, and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3 (counting from the 5' end); and (b) an antisense strand having: (i) 21 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end), and (iii) containing phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end); The RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0474] In certain embodiments, the iRNA used in the methods of the invention is an agent selected from an agent selected from any one of Tables 2-3. These agents may further comprise a ligand.

[0475] III. Ligand-Conjugated iRNA Another modification of the iRNA of the invention involves chemically linking the iRNA to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA, for example, into cells. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556).In other embodiments, the ligand is selected from the group consisting of cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0476] In certain embodiments, a ligand alters the distribution, targeting, or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a compartment of a cell or organ, a tissue, an organ, or a region of the body, e.g., compared to a species in which such ligand is absent. In some embodiments, a ligand does not participate in double-strand pairing in a double-stranded nucleic acid.

[0477] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0478] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody, that binds to a specific cell type, e.g., a kidney cell. The targeting group can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, e.g., N-acetyl-galactosamine.

[0479] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Examples of suitable nucleotides include cholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0480] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to specific cell types such as hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0481] The ligand can be a substance, e.g., a drug, that can increase uptake of the iRNA agent into the cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, e.g., taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0482] In some embodiments, the ligands that bind to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0483] The ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing reactive functional pendant side chains, such as those resulting from the attachment of a linking molecule onto the oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing a linking moiety attached thereto.

[0484] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors, such as Applied Biosystems® (Foster City, Calif.). Any other method for such synthesis known in the art may additionally or alternatively be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0485] In the ligand-conjugated iRNAs and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or conjugated nucleotide or nucleoside precursors already bearing a binding moiety, or conjugated ligand-nucleotide or ligand-nucleoside precursors already bearing a ligand molecule, or on non-nucleoside ligand-bearing building blocks.

[0486] When a nucleotide-conjugate precursor already bearing a linking moiety is used, synthesis of the sequence-specifically linked nucleoside is typically completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0487] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. In one embodiment, such a lipid or lipid-based molecule binds a serum protein, such as human serum albumin (HSA). The HSA-binding ligand allows the conjugate to be distributed to a target tissue in the body, for example, a target tissue other than the kidney. For example, the target tissue can be the liver, including the parenchymal cells of the liver. Other molecules capable of binding HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport into target cells or cell membranes, or (c) modulate binding to serum proteins, such as HSA.

[0488] Lipid-based ligand can be used to inhibit, for example, control, the binding of conjugate to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to target to kidney, and therefore is less likely to be eliminated from the body.The lipid or lipid-based ligand that binds less strongly to HSA can be used so that the conjugate targets to kidney.

[0489] In certain embodiments, the lipid-based ligand binds HSA. In one embodiment, it binds HSA with sufficient affinity so that the conjugate is distributed to non-renal tissues. However, the affinity is preferably not so strong that the HSA-ligand binding is irreversible.

[0490] In other embodiments, the lipid-based ligand binds HSA weakly or not at all. In one embodiment, the conjugate is distributed to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0491] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, e.g., hepatocytes. Also included are HSA and low-density lipoprotein (LDL).

[0492] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In one embodiment, the cell-penetrating agent is amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. In one embodiment, the helical agent is an alpha-helical agent having a lipophilic phase and a lipophobic phase.

[0493] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0494] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can include a hydrophobic membrane translocating sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 15)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and cell membrane-spanning proteins. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 16)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 17)) have been found to be capable of functioning as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, e.g., from phage display libraries or one-bead-one compound (OBOC) combinatorial libraries (Lam et al. al., Nature, 354:82-84, 1991). Examples of peptides or peptidomimetics tethered to dsRNA agents via incorporated monomer units for cell targeting purposes include arginine-glycine-aspartic acid (RGD)-peptides or RGD mimetics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, e.g., to increase stability or to affect conformational properties. Any of the structural modifications described below can be utilized.

[0495] RGD peptides for use in the compositions and methods of the present invention can be linear or cyclic and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptidiomimetics can include D-amino acids as well as synthetic RGD mimetics. In addition to RGD, other moieties can be used that target integrin ligands, such as PECAM-1 or VEGF.

[0496] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0497] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids, as described herein, and are suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself (which may be linear, branched, or cyclic) composed of one or more monosaccharide units having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound having as part thereof a carbohydrate moiety (which may be linear, branched, or cyclic) composed of one or more monosaccharide units, each having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Exemplary carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides consisting of about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide resins. Particular monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars, and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0498] In certain embodiments, the carbohydrate conjugates used in the compositions and methods of the present invention are monosaccharides.

[0499] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US Pat. No. 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate functions as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by functioning as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0500] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., via a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to an iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to an iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., as described herein.

[0501] In certain embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a tetravalen...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a pharma- ceutically acceptable salt thereof for inhibiting expression of retinal binding protein 4 (RBP4) in a cell, the dsRNA agent comprising a sense strand and an antisense strand forming a double-stranded region; (a) the sense strand comprises at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:2 by no more than 3 nucleotides; or (b) the antisense strand comprises a region complementary to an mRNA encoding RBP4, the region of complementarity being selected from the group consisting of those shown in Tables 1 to 2 below. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 1-9】 【Table 1-10】 【Table 1-11】 【Table 1-12】 【Table 1-13】 【Table 1-14】 【Table 1-15】 【Table 1-16】 【Table 1-17】 【Table 1-18】 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2-5】 【Table 2-6】 【Table 2-7】 【Table 2-8】 【Table 2-9】 【Table 2-10】 【Table 2-11】 【Table 2-12】 【Table 2-13】 【Table 2-14】 【Table 2-15】 【Table 2-16】 【Table 2-17】 【Table 2-18】 【Table 2-19】 【Table 2-20】 【Table 2-21】 comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense nucleotide sequences in any one of A double-stranded ribonucleic acid (dsRNA) agent or a pharma- ceutically acceptable salt thereof.

2. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said dsRNA agent comprises at least one nucleotide modification.

3. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein all of the nucleotides of the sense strand comprise a nucleotide modification, all of the nucleotides of the antisense strand comprise a nucleotide modification, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a nucleotide modification.

4. At least one of the nucleotide modifications is a deoxy-nucleotide modification, a 3'-terminal deoxythymidine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy nucleotide modification, a locked nucleotide modification, a non-locked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2'-amino nucleotide modification, a 2'-O-allyl nucleotide modification, a 2'-C-alkyl nucleotide modification, a 2'-hydroxyl nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl nucleotide modification, a morpholino nucleotide modification, a phosphatase inhibitor ...

3. The dsRNA agent of claim 2, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of a phos- amidate modification, a non-natural base modification comprising a nucleotide, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexenyl nucleotide modification, a nucleotide modification comprising a phosphorothioate group, a nucleotide modification comprising a methylphosphonate group, a nucleotide modification comprising a 5'-phosphate, a nucleotide modification comprising a 5'-phosphate mimic, a thermo- destabilized nucleotide modification, a glycol nucleic acid (GNA) modification, a nucleotide modification comprising a 2' phosphate, and a 2-O-(N-methylacetamido) nucleotide modification, and combinations thereof.

5. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the double-stranded region is 19-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 23-27 nucleotide pairs in length, or 21-23 nucleotide pairs in length.

6. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein each strand is independently 30 nucleotides or less in length.

7. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

8. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide or at least 2 nucleotides.

9. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, further comprising a ligand.

10. 10. The dsRNA agent of claim 9, or a pharma- ceutically acceptable salt thereof, wherein said ligand is conjugated to the 3' end of a sense strand of said dsRNA agent.

11. 10. The dsRNA agent of claim 9, or a pharma- ceutically acceptable salt thereof, wherein said ligand is an N-acetylgalactosamine (GalNAc) derivative.

12. 10. The dsRNA agent of claim 9, or a pharma- ceutically acceptable salt thereof, wherein said ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

13. 12. The dsRNA agent of claim 11, or a pharma- ceutically acceptable salt thereof, wherein said ligand is: 【Chemistry 1】

14. The dsRNA agent is conjugated to the ligand as shown in the following schematic diagram: 【Chemistry 2】 14. The dsRNA agent of claim 13, wherein X is O or S, or a pharma- ceutically acceptable salt thereof.

15. 2. The dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, wherein said dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

16. 13. A cell comprising the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof.

17. 10. A pharmaceutical composition for inhibiting expression of a gene encoding retinal binding protein 4 (RBP4), comprising the dsRNA agent of claim 1 or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

18. 20. The pharmaceutical composition of claim 17, wherein the dsRNA agent, or a pharma- ceutically acceptable salt thereof, is in an unbuffered solution.

19. 19. The pharmaceutical composition of claim 18, wherein the non-buffered solution is saline or water.

20. 20. The pharmaceutical composition of claim 17, wherein the dsRNA agent, or a pharma- ceutically acceptable salt thereof, is in a buffered solution.

21. 21. The pharmaceutical composition of claim 20, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

22. 22. The pharmaceutical composition of claim 21, wherein the buffer solution is phosphate buffered saline (PBS).

23. 18. An in vitro method of inhibiting expression of the retinal binding protein 4 (RBP4) gene in a cell, comprising contacting the cell with a dsRNA agent or a pharma- ceutically acceptable salt thereof of claim 1, or a pharmaceutical composition of claim 17, thereby inhibiting expression of the RBP4 gene in the cell.

24. 24. The method of claim 23, wherein contacting the cell with the dsRNA agent or a pharma- ceutically acceptable salt thereof inhibits expression of RBP4 by at least 50%, 60%, 70%, 80%, 90%, or 95%.

25. 11. A pharmaceutical composition for treating a subject having a disorder that would benefit from a decrease in retinal binding protein 4 (RBP4) expression, comprising a therapeutically effective amount of the dsRNA agent of claim 1 or a pharma- ceutical acceptable salt thereof.

26. 11. A pharmaceutical composition for preventing at least one symptom in a subject having a disorder that would benefit from a decrease in retinal binding protein 4 (RBP4) expression, comprising a therapeutically effective amount of the dsRNA agent of claim 1 or a pharma- ceutical acceptable salt thereof.

27. 27. The pharmaceutical composition of claim 25 or 26, wherein the disorder is an RBP4-associated disorder.

28. 28. The pharmaceutical composition of claim 27, wherein the RBP4-associated disorder is an ophthalmic disease selected from the group consisting of Stargardt's disease, diabetic retinopathy, wet macular degeneration, dry macular degeneration, iris coloboma, comedonal acne syndrome, microphthalmia, basement membrane drusen, diabetic macular edema, and retinal vein occlusion.

29. 29. The pharmaceutical composition of claim 28, wherein the RBP4-associated disorder is Stargardt's disease.

30. 29. The pharmaceutical composition of claim 28, wherein the RBP4-associated disorder is diabetic retinopathy.

31. 29. The pharmaceutical composition of claim 28, wherein the RBP4-associated disorder is wet macular degeneration.

32. 29. The pharmaceutical composition of claim 28, wherein the RBP4-associated disorder is dry macular degeneration.

33. 29. The pharmaceutical composition of claim 28, wherein the RBP4-associated disorder is a metabolic disorder selected from the group consisting of disorders of glucose and lipid homeostasis and cardiovascular diseases.

34. 34. The pharmaceutical composition of claim 33, wherein the metabolic disorder is insulin resistance associated with type II diabetes.

35. 27. The pharmaceutical composition of claim 25 or 26, wherein the subject is a human.

36. 27. The pharmaceutical composition according to claim 25 or 26, wherein the pharmaceutical composition is for subcutaneous and / or intravitreal administration.

37. 27. The pharmaceutical composition of claim 25 or 26, further comprising an additional therapeutic agent for the treatment of an RBP4-associated disorder.

38. 38. The pharmaceutical composition of claim 37, wherein the additional therapeutic agent is selected from the group consisting of agents that inhibit transthyretin (TTR) expression and / or activity, the synthetic retinoid fenretinide, anti-VEGF therapy, corticosteroids, insulin, glucagon-like peptide 1 agonists, sulfonylureas, seglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, and combinations of any of the foregoing.

39. 18. A kit, vial, or syringe comprising the dsRNA agent of claim 1, or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of claim 17.

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