Transthyretin (TTR) iRNA Compositions and Methods of Use Thereof

Abstract

The present invention relates to RNAi agents, such as double-stranded RNA (dsRNA) agents that target the transthyretin (TTR) gene. The present invention also relates to methods of using such RNAi agents to inhibit expression of the TTR gene, and to methods of preventing and treating TTR-related disorders, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 228,830, filed August 3, 2021, the entire contents of which are incorporated herein by reference.

[0002] Background of the Invention Transthyretin (TTR), also known as prealbumin, is found in serum and cerebrospinal fluid (CSF). TTR transports retinol-binding protein (RBP) and thyroxine (T4) and also acts as a carrier of retinol (vitamin A) through its association with RBP in blood and CSF. Transthyretin is named after its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apoA-I (the major HDL apolipoprotein), amyloid β-peptide, and neuropeptide Y. See Liz, MA et al. (2010) IUBMB Life, 62(6):429-435.

[0003] 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 link the monomers into dimers. A short loop from each monomer forms the major dimer-dimer interaction. Two pairs of these loops separate the opposing convex beta-sheets of the dimer, forming an internal channel.

[0004] The liver is the major site of TTR expression. Other important sites of expression include the choroid plexus, retina (particularly the retinal pigment epithelium), and pancreas.

[0005] Transthyretin is one of at least 27 distinct types of proteins that are precursor proteins in the formation of amyloid fibrils. See Guan, J. et al. (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi:10.1152 / ajpheart.00815.2011. Extracellular deposition of amyloid fibrils in organs and tissues is a hallmark of amyloidosis. Amyloid fibrils are composed of misfolded protein aggregates, which can result from either overproduction of the precursor protein or specific mutations in the precursor protein. The amyloidogenic potential of TTR may be related to its extensive beta-sheet structure. X-ray crystallographic studies have shown that certain amyloidogenic mutations destabilize the tetrameric structure of the protein. See, for example, Saraiva MJM (2002) Expert Reviews in Molecular Medicine, 4(12):1-11.

[0006] Amyloidosis is a general term for a group of amyloid diseases characterized by amyloid deposits. Amyloid diseases are classified based on their precursor proteins, e.g., names begin with the letter "A" for amyloid followed by the abbreviation for the precursor protein, e.g., ATTR for amyloidogenic transthyretin (Id.).

[0007] There are numerous TTR-related diseases, most of which are amyloid diseases. Normal-sequence TTR is associated with cardiac amyloidosis in elderly individuals, referred to as senile systemic amyloidosis (SSA) (also referred to as senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA is often accompanied by microscopic deposits in many other organs. TTR amyloidosis manifests in various forms. When the peripheral nervous system is more prominently affected, the disease is called familial amyloid polyneuropathy (FAP). When the heart is primarily involved but not the nervous system, the disease is called familial amyloid cardiomyopathy (FAC). The third major type of TTR amyloidosis is leptomeningeal amyloidosis, also known as leptomeningeal or meningeal cerebrovascular amyloidosis, central nervous system (CNS) amyloidosis, or amyloidosis type VII. Mutations in TTR can also cause amyloid vitreous opacities, carpal tunnel syndrome, and euthyroid hyperthyroxinemia, non-amyloid diseases thought to be secondary to increased association of thyroxine with TTR due to mutant TTR molecules with increased affinity for thyroxine. See, e.g., Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.

[0008] Abnormal TTR alleles can be inherited or acquired through somatic mutations. Guan, J. et al. (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi:10.1152 / ajpheart.00815.2011. Transthyretin-associated ATTR is the most common form of hereditary systemic amyloidosis (Lobato, L. (2003) J. Nephrol., 16:438-442). TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of ATTR. More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis. TTR mutations usually result in systemic amyloid deposition, particularly involving the peripheral nervous system, although some mutations are associated with cardiomyopathy or vitreous opacities. (same document)

[0009] The V30M mutation is the most common TTR mutation. See, e.g., Lobato, L. (2003) J Nephrol, 16:438-442. The V122I mutation is carried by 3.9% of the African American population and is the most common cause of FAC. Jacobson, DR et al. (1997) N. Engl. J. Med. 336 (7): 466-73. SSA is estimated to affect more than 25% of the population over 80 years of age. Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. USA 87 (7): 2843-5.

[0010] Additional TTR-related diseases include ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD) such as dry AMD and wet AMD, metabolic disorders such as disorders of glucose and lipid homeostasis such as insulin resistance associated with type II diabetes, and cardiovascular disease.

[0011] For example, the primary pathological abnormality in Stargardt disease, a genetic eye disorder, is the excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) coincides with an age-dependent increase in the incidence of atrophic (dry) age-related macular degeneration (AMD) and is therefore an important pathogenic factor contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation to the RPE, which requires the formation of a tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in serum. Knockdown of TTR, which regulates RBP4, has been shown to significantly reduce both RBP4 levels and the amount of retinol delivered to target tissues, thereby inhibiting the formation of lipofuscin bisretinoids.

[0012] Furthermore, elevated RBP4 in the circulation of type 2 diabetic patients 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 in normal mice or injection of human RBP4 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 of circulating RBP4 or RBP4 expression levels with established cardiovascular disease (CVD) risk factors, including metabolic syndrome, systemic / central obesity, dyslipidemia, inflammatory markers, and hypertension, have been documented (Qi Q, et al., J Clin Endocrinol Metab. 2007; 92:4827-4834; Ingelsson E, et al., Atherosclerosis. 2009; 206:239-244). As mentioned above, TTR regulates RBP4, and thus, a decrease in TTR results in a decrease in RBP4.

[0013] Currently available treatments for TTR-related diseases, such as ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), including dry AMD and wet AMD, metabolic disorders such as impaired glucose and lipid homeostasis, including insulin resistance associated with type II diabetes, and cardiovascular disease, are not completely effective and can cause serous side effects such as conjunctival hemorrhage, increased intraocular pressure, infection, retinal detachment, and ophthalmitis. Similarly, current treatments for metabolic diseases, including diet, exercise, and drugs to help control blood pressure, cholesterol, and blood sugar levels, are not always effective.

[0014] Thus, there is a need in the art for effective treatments for TTR-related diseases. Summary of the Invention

[0015] The present invention provides iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of a gene encoding transthyretin (TTR). The TTR gene can be in a cell, e.g., in a cell within 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 TTR gene and / or to treat subjects who would benefit from inhibition or reduction of expression of the TTR gene, e.g., subjects suffering from or susceptible to a TTR-related disorder, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, metabolic disorders, e.g., disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, and cardiovascular disease.

[0016] Thus, in one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of transthyretin (TTR) 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, 20, or 21 contiguous nucleotides that differ by no more than 3, e.g., 3, 2, 1, or 0 nucleotides, from the nucleotide sequence 5'-AGAGTAUUCCAUUUUUACU-3', and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3, e.g., 3, 2, 1, or 0 nucleotides, from the nucleotide sequence 5'-AGUAAAAAUGGAAUACUCUUG-3'. comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides that differ by 0 nucleotides, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a nucleotide modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the sense strand comprises at least two 2'-fluoro modifications and the antisense strand comprises at least two 2'-fluoro modifications, the sense strand comprises two phosphorothioate linkages and the antisense strand comprises four phosphorothioate linkages, and at least one strand is conjugated to a ligand.

[0017] In one embodiment, the sense strand contains two to six 2'-fluoro modifications.

[0018] In one embodiment, the sense strand contains four 2'-fluoro modifications.

[0019] In one embodiment, the four 2'-fluoro modifications are located at positions 7 and 9-11 from the 5' end of the sense strand.

[0020] In one embodiment, the sense strand comprises three 2'-fluoro modifications.

[0021] In one embodiment, the three 2'-fluoro modifications are located at positions 7-9 from the 5' end of the sense strand.

[0022] In one embodiment, the antisense strand contains two to six 2'-fluoro modifications.

[0023] In one embodiment, the antisense strand contains four 2'-fluoro modifications.

[0024] In one embodiment, the four 2'-fluoro modifications are located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand.

[0025] In one embodiment, the antisense strand comprises two 2'-fluoro modifications.

[0026] In one embodiment, the two 2'-fluoro modifications are located at positions 14 and 16 from the 5' end of the antisense strand.

[0027] In one embodiment, the sense strand comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at the 5' end.

[0028] In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at the 3' end and two phosphorothioate internucleotide linkages between the three terminal nucleotides at the 5' end.

[0029] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0030] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0031] In one embodiment, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0032] In one embodiment, the ligand is: [ka]

[0033] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] wherein X is O or S.

[0034] In one embodiment, X is O.

[0035] In one embodiment, the double-stranded region is 19-30 nucleotide pairs in length.

[0036] In one embodiment, the double-stranded region is 19 to 25 nucleotide pairs in length.

[0037] In one embodiment, the double-stranded region is 19 to 23 nucleotide pairs in length.

[0038] In one embodiment, the double-stranded region is 23-27 nucleotide pairs in length.

[0039] In one embodiment, the double-stranded region is 21-23 nucleotide pairs in length.

[0040] In one embodiment, each strand is independently 30 nucleotides or less in length.

[0041] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0042] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0043] In one embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0044] In one embodiment, the sense strand comprises the nucleotide sequence 5'-CAAGAGUAUUCCAUUUUUACU-3' and the antisense strand comprises the nucleotide sequence 5'-AGUAAAAAUGGAAUACUCUUGGU-3'.

[0045] In one embodiment, the sense strand comprises the nucleotide sequence 5'-CCAAGAGUAUUCCAUUUUUAU-3' and the antisense strand comprises the nucleotide sequence 5'-AUAAAAAUGGAAUACUCUUGGUU-3'.

[0046] In one embodiment, the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3', and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3', 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; and s is a phosphorothioate linkage.

[0047] In one embodiment, the nucleotide sequence of the sense strand differs by no more than three bases from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' and the nucleotide sequence of the antisense strand differs by no more than three bases from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3'.

[0048] In one embodiment, the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3'.

[0049] In one embodiment, the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3'.

[0050] In one embodiment, the sense strand comprises the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' and the antisense strand comprises the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3'.

[0051] In one embodiment, the sense strand consists of the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' and the antisense strand consists of the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3'.

[0052] In one embodiment, the sense strand comprises the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacuL96-3' and the antisense strand comprises the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3', where L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0053] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] wherein X is O.

[0054] In one embodiment, the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-cscsaagaGfuAfUfUfccauuuuuau-3', and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-asUfsaaaAfauggaauAfcUfcuuggsusu-3', 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, and s is a phosphorothioate linkage.

[0055] In one embodiment, the nucleotide sequence of the sense strand differs by no more than three bases from the nucleotide sequence 5'-cscsaagaGfuAfUfUfccauuuuuau-3' and the nucleotide sequence of the antisense strand differs by no more than three bases from the nucleotide sequence 5'-asUfsaaaAfauggaauAfcUfcuuggsusu-3'.

[0056] In one embodiment, the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-cscsaagaGfuAfUfUfccauuuuuau-3' and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-asUfsaaaAfauggaauAfcUfcuuggsusu-3'.

[0057] In one embodiment, the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-cscsaagaGfuAfUfUfccauuuuuau-3' and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-asUfsaaaAfauggaauAfcUfcuuggsusu-3'.

[0058] In one embodiment, the sense strand comprises the nucleotide sequence 5'-cscsaagaGfuAfUfUfccauuuuuau-3' and the antisense strand comprises the nucleotide sequence 5'-asUfsaaaAfauggaauAfcUfcuuggsusu-3'.

[0059] In one embodiment, the sense strand consists of the nucleotide sequence 5'-cscsaagaGfuAfUfUfccauuuuuau-3' and the antisense strand consists of the nucleotide sequence 5'-asUfsaaaAfauggaauAfcUfcuuggsusu-3'.

[0060] In one embodiment, the sense strand comprises the nucleotide sequence 5'-cscsaagaGfuAfUfUfccauuuuuau-3', the antisense strand comprises the nucleotide sequence 5'-asUfsaaaAfauggaauAfcUfcuuggsusu-3', and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0061] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] wherein X is O.

[0062] In one embodiment, the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-asgsagdTaUfUfCfcauuuuuacu-3', and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-asdGsuadAadAauggaaUfaCfucususg-3', 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; dT is 2'-deoxythymidine-3'-phosphate; dG is 2'-deoxyguanosine-3'-phosphate; dA is 2'-deoxyadenosine-3'-phosphate; and s is a phosphorothioate linkage.

[0063] In one embodiment, the nucleotide sequence of the sense strand differs by no more than three bases from the nucleotide sequence 5'-asgsagdTaUfUfCfcauuuuuacu-3' and the nucleotide sequence of the antisense strand differs by no more than three bases from the nucleotide sequence 5'-asdGsuadAadAauggaaUfaCfucususg-3'.

[0064] In one embodiment, the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-asgsagdTaUfUfCfcauuuuuacu-3' and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-asdGsuadAadAauggaaUfaCfucususg-3'.

[0065] In one embodiment, the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-asgsagdTaUfUfCfcauuuuuacu-3' and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-asdGsuadAadAauggaaUfaCfucususg-3'.

[0066] In one embodiment, the sense strand comprises the nucleotide sequence 5'-asgsagdTaUfUfCfcauuuuuacu-3' and the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-asdGsuadAadAauggaaUfaCfucususg-3'.

[0067] In one embodiment, the sense strand consists of the nucleotide sequence 5'-asgsagdTaUfUfCfcauuuuuacu-3', and the nucleotide sequence of the antisense strand consists of the nucleotide sequence 5'-asdGsuadAadAauggaaUfaCfucususg-3'.

[0068] In one embodiment, the sense strand comprises the nucleotide sequence 5'-asgsagdTaUfUfCfcauuuuuacu-3', the nucleotide sequence of the antisense strand comprises the nucleotide sequence 5'-asdGsuadAadAauggaaUfaCfucususg-3', and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0069] In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the diagram below: [ka] wherein X is O.

[0070] The invention also provides pharmaceutical compositions comprising a cell and, eg, a pharmaceutically acceptable carrier, that include a dsRNA agent of the invention.

[0071] In one embodiment, a pharmaceutical composition of the invention comprises a non-buffered solution, such as saline or water.

[0072] In one embodiment, the pharmaceutical composition of the invention comprises a buffer solution, such as a solution comprising acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS).

[0073] In one aspect, the invention provides a method for inhibiting expression of the transthyretin (TTR) gene in a cell, the method comprising contacting the cell with a dsRNA agent of the invention or with a pharmaceutical composition of the invention, thereby inhibiting expression of the TTR gene in the cell.

[0074] In one embodiment, the cell is in a subject.

[0075] In one embodiment, the subject is a human.

[0076] In one embodiment, the subject is a subject suffering from a TTR-related disease.

[0077] In one embodiment, the subject is at risk of developing a TTR-related disease.

[0078] In one embodiment, the subject carries a TTR gene mutation associated with the development of a TTR-related disorder.

[0079] In one embodiment, the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD) such as dry AMD and wet AMD, metabolic disorders such as disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, and cardiovascular diseases.

[0080] In one embodiment, the subject has TTR-related amyloidosis, and the method reduces amyloid TTR deposits in the subject.

[0081] In one embodiment, contacting a cell with a dsRNA agent inhibits expression of TTR by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0082] In one embodiment, inhibiting expression of TTR reduces TTR protein levels in the subject's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0083] In another aspect, the present invention provides a method for treating a subject suffering from or at risk of developing a TTR-related disorder, comprising administering to the subject a therapeutically or prophylactically effective amount of a double-stranded RNAi agent of the present invention or a pharmaceutical composition of the present invention, thereby treating the subject.

[0084] In one embodiment, the subject is a human.

[0085] In one embodiment, the subject is a subject suffering from a TTR-related disease.

[0086] In one embodiment, the subject is at risk of developing a TTR-related disease.

[0087] In one embodiment, the subject carries a TTR gene mutation associated with the development of a TTR-related disorder.

[0088] In one embodiment, the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD) such as dry AMD and wet AMD, metabolic disorders such as disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, and cardiovascular diseases.

[0089] In one embodiment, the subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the method reduces amyloid TTR deposits in the subject.

[0090] In one embodiment, the ATTR is a hereditary ATTR (h-ATTR).

[0091] In one embodiment, the ATTR is a non-hereditary ATTR (wt ATTR).

[0092] In one embodiment, administration of the dsRNA agent or pharmaceutical composition to a subject improves at least one indicator of neurological disorder, quality of life, ongoing neurological damage, or cardiovascular disorder in the subject.

[0093] In one embodiment, the double-stranded RNAi agent is administered to a human subject by subcutaneous or intravenous administration.

[0094] In one embodiment, the subcutaneous administration is self-administration.

[0095] In one embodiment, self-administration is via a pre-filled syringe or an auto-injector syringe.

[0096] In one embodiment, the method further comprises assessing the level of TTR mRNA expression or TTR protein expression in a sample from the human subject.

[0097] In one aspect, the invention provides a kit comprising a dsRNA agent of the invention, or a pharmaceutical composition of the invention, and, optionally, instructions for use.

[0098] In another aspect, the invention provides a vial containing a dsRNA agent of the invention, or a pharmaceutical composition of the invention.

[0099] In one aspect, the invention provides a syringe comprising a dsRNA agent of the invention, or a pharmaceutical composition of the invention.

[0100] In one aspect, the invention provides an RNA-induced silencing complex (RISC) comprising the antisense strand of a dsRNA agent of the invention. [Brief explanation of the drawings]

[0101] [Figure 1] Figure 1 is a graph showing human TTR mRNA levels in mice (n=3 per group) subcutaneously administered a single 1 mg / kg dose of the indicated dsRNA duplex on day 7. Human TTR mRNA levels are shown relative to control levels detected with PBS treatment. [Figure 2]FIG. 2 is a graph showing the effect of administering a single subcutaneous dose of 0.5 mg / kg of AD-649263, AD-1134938, AD-649264, AD-1134966, or AD-65496 on TTR mRNA levels in non-human primates. DETAILED DESCRIPTION OF THE INVENTION

[0102] The present invention provides iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the transthyretin (TTR) 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 (TTR) in a mammal.

[0103] The iRNAs of the present invention are designed to target the human transthyretin (TTR) gene, including portions of the gene conserved in TTR orthologs of other mammalian species. Without intending to be limited by theory, it is believed that the combination of specific target sites in these iRNAs, specific modifications, and ligand binding improves the efficacy, stability, potency, durability, and safety of the iRNAs of the present invention. Another unexpected and highly desirable feature of the iRNAs of the present invention is that they exhibit no or very low off-target activity. In the examples presented herein, the inventors have identified dsRNA agents of the present invention that exhibit the lowest observed off-target activity of any dsRNA agent targeting any gene to date.

[0104] The present invention also provides methods for treating and preventing transthyretin (TTR)-related disorders, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, ocular diseases, such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), such as dry AMD and wet AMD, metabolic disorders, such as disorders of glucose and lipid homeostasis, such as insulin resistance associated with type II diabetes, and cardiovascular disease, using iRNA compositions that result in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the TTR gene.

[0105] The iRNA of the present invention comprises an RNA strand (antisense strand) having a region that is up to about 30 nucleotides in length or less, 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 TTR gene.

[0106] 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 TTR 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.

[0107] The use of iRNA of the present invention allows the targeted degradation of mRNA of the corresponding gene (TTR gene) in mammals.The inventors have demonstrated through in vitro assay that iRNA targeting TTR gene can strongly mediate RNAi, resulting in significant inhibition of the expression of TTR gene.Therefore, the method and composition comprising these iRNA are useful for treating subjects with TTR-related disorders, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, eye diseases, such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), such as dry AMD and wet AMD, metabolic disorders, such as glucose and lipid homeostasis disorders, such as insulin resistance associated with type II diabetes, or cardiovascular disease.

[0108] Thus, the present invention provides methods and combination therapies for treating subjects with disorders that would benefit from inhibiting or reducing expression of the TTR gene, e.g., transthyretin (TTR)-associated disorders, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, ocular diseases, e.g., Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, metabolic disorders, e.g., disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, and cardiovascular disease, using iRNA compositions that result in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the TTR gene.

[0109] The present invention also provides methods for preventing at least one symptom in a subject having a transthyretin (TTR)-associated disorder that would benefit from inhibiting or reducing expression of the TTR gene, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD) such as dry AMD and wet AMD, metabolic disorders such as disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, and cardiovascular disease.

[0110] The following detailed description of the invention discloses methods of making and using compositions containing iRNAs that inhibit expression of the TTR gene, as well as compositions, uses, and methods of treating subjects who would benefit from inhibition and / or reduction of expression of the TTR gene, e.g., subjects susceptible to or diagnosed with a TTR-related disorder.

[0111] 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.

[0112] 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.

[0113] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0114] 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."

[0115] 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.

[0116] The terms "at least," "greater than," or "or more" preceding a number or series of numbers, when clear from the context, are understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included. 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.

[0117] As used herein, "less than" or "or less than" refers to the value adjacent to the phrase and, if logical from the context, to zero, any logically smaller value or integer than the value.For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides.When "less than" is used before a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.As used herein, a range includes both upper and lower limits.

[0118] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0119] In the event of a conflict between a given target site and the nucleotide sequence for either the sense or antisense strand, the given sequence controls.

[0120] 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.

[0121] As used herein, "transthyretin" ("TTR") refers to the well-known gene and protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter of retinol-binding protein (RBP), thyroxine (T4), and retinol, and also acts as a protease. The liver secretes TTR into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR is also expressed in the pancreas and retinal pigment epithelium. The greatest clinical relevance of TTR is that both normal (wild-type) and mutant TTR proteins can form amyloid fibrils, which aggregate into extracellular deposits and cause amyloidosis. For a review, see, e.g., Saraiva MJM (2002) Expert Reviews in Molecular Medicine, 4(12):1-11. The molecular cloning and nucleotide sequence of rat transthyretin, as well as the distribution of mRNA expression, are described in Dickson, PW et al. (1985) J. Biol. Chem. 260(13)8214-8219. The X-ray crystal structure of human TTR is described in Blake, CC et al. (1974) J Mol Biol 88, 1-12.

[0122] The sequence of human TTR mRNA transcript can be found in National Center for Biotechnology Information (NCBI) RefSeq Accession No. NM_000371.4 (SEQ ID NO: 1, reverse complement, SEQ ID NO: 5). The sequence of mouse TTR mRNA can be found in RefSeq Accession No. NM_013697.2 (SEQ ID NO: 2, reverse complement, SEQ ID NO: 6). The sequence of rat TTR mRNA can be found in RefSeq Accession No. NM_012681.1 (SEQ ID NO: 3, reverse complement, SEQ ID NO: 7). The sequence of cynomolgus monkey (Macaca fascicularis) TTR mRNA can be found in RefSeq Accession No. NM_001283593.1 (SEQ ID NO: 4, reverse complement, SEQ ID NO: 8). The sequence of rhesus monkey (Macaca mulatta) TTR mRNA can be found in RefSeq accession number NM_001261679.1 (SEQ ID NO: 9, reverse complement SEQ ID NO: 10).

[0123] Further examples of TTR mRNA sequences are readily available through public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0124] Further information regarding TTR can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=TTR.

[0125] 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.

[0126] The term TTR, as used herein, also refers to variations of the TTR gene, including variants provided in SNP databases. Numerous sequence variations within the TTR gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?term=TTR, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0127] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the TTR gene, e.g., an 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 the mRNA molecule formed during transcription of the TTR gene.

[0128] 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.

[0129] 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.

[0130] Generally, "G", "C", "A", "T" and "U" respectively represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as base.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 that contains the nucleotide with such replacement moieties.For example, but not limited to, the nucleotide that contains inosine as its base can base pair with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be substituted with, for example, the nucleotide that contains inosine in the nucleotide sequence of the dsRNA of the present invention. In another example, adenine and cytosine anywhere within an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured herein.

[0131] 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 TTR gene within cells, e.g., within hepatocytes in a subject, such as a mammalian subject.

[0132] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, for example, a TTR target mRNA sequence, and mediates 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., TTR gene.Therefore, the term " siRNA " is also used herein to refer to the iRNA described above.

[0133] In certain embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNA is generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded siRNAs are 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 the single-stranded siRNA described herein or as the single-stranded siRNA that is chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0134] In certain embodiments, the "iRNA" used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNA agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure, comprising two antiparallel, substantially complementary nucleic acid strands, which are referred to as having a "sense" or "antisense" orientation with respect to the target RNA, i.e., the TTR gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, for example, mRNA, via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0135] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as described in detail herein, each strand 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 linkage, or a modified nucleobase, or any combination thereof.Therefore, the term modified nucleotide includes the substitution, addition, or removal of, for example, functional groups or atoms, to the internucleoside linkage, 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.

[0136] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0137] The duplex 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 duplex region may range in length from 19 to 21 base pairs, e.g., 21 base pairs in length, 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 length. In certain embodiments, the duplex 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.

[0138] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a 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, 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.

[0139] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, they can, but do not necessarily, 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 5'-end of the corresponding strand that forms a duplex structure, the connecting structure is called a "linker." The RNA strands can have the same or different number 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 duplex structure, RNAi can also include one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes 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, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0140] 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 TTR gene, and mediates cleavage of the target RNA.

[0141] In some embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a TTR target mRNA sequence, and mediates cleavage of the target RNA.

[0142] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can contain at least one nucleotide overhang, or the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, a certain overhanging nucleotide can be present on the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0143] In one embodiment, the antisense strand of dsRNA has an overhang of 1-10 nucleotides at 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.In one embodiment, the sense strand of dsRNA has an overhang of 1-10 nucleotides at 3'-end 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 replaced with nucleoside thiophosphate.

[0144] In certain embodiments, the antisense strand of dsRNA has 1-10 nucleotides at 3'-end or 5'-end, for example, 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides overhang.In one embodiment, the sense strand of dsRNA has 1-10 nucleotides at 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides overhang.In another embodiment, one or more nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0145] 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., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In certain embodiments, the overhang on the sense strand, the antisense strand, or both strands can comprise an extended length greater 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.

[0146] " Blunt " or " blunt end " means that there is no unpaired nucleotide at the end of a double-stranded RNA agent, i.e., there is no nucleotide overhang.A " blunt-ended " double-stranded RNA agent is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule.The RNAi agent of the present invention includes an RNAi agent that does not have a nucleotide overhang at one end (i.e., an agent that has one overhang and one blunt end), or an RNAi agent that does not have a nucleotide overhang at either end.In most cases, these molecules will be double-stranded throughout their entire length.

[0147] 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., TTR mRNA.

[0148] 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., a TTR nucleotide sequence. If the region of complementarity is not perfectly complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most tolerable mismatch is in the terminal region, 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 present invention contains a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of a double-stranded RNA agent of the present 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 present 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 present invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA agent. In other embodiments, the nucleotide mismatch is, for example, within the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0149] Therefore, 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 be optionally limited to be within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to a region of the TTR gene generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting the expression of the TTR gene. It is important to consider the efficacy of mismatched RNAi agents in inhibiting TTR gene expression, especially when specific complementary regions in the TTR gene are known to have polymorphic sequence variation within the population.

[0150] The term "sense strand" or "passenger strand," as used herein, refers 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.

[0151] 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.

[0152] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site.Cleavage site is the site on the target where cleavage occurs.In some embodiments, the cleavage region comprises three bases on either end of the cleavage site and directly adjacent to the cleavage site.In some embodiments, the cleavage region comprises two bases on either end of the cleavage site and directly adjacent to the cleavage site.In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and this cleavage region comprises nucleotides 11, 12, and 13.

[0153] As used herein, unless otherwise specified, 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 duplex 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 may 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, may 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.

[0154] A complementary sequence in an iRNA, such as in a dsRNA described herein, includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs during hybridization for a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its final application, for example, in vitro or in vivo, inhibiting gene expression. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs shall not be considered mismatches when 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.

[0155] "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, including, 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.

[0156] The terms "complementary," "fully complementary," and "substantially complementary" 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.

[0157] 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 TTR gene). For example, a polynucleotide is complementary to at least a portion of a TTR mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the TTR gene.

[0158] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target TTR sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target TTR sequence, and comprise a contiguous nucleotide sequence that is at least 80% complementary, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary 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.

[0159] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target TTR 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, 5, and 6, or to a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 5, and 6.

[0160] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide and thus identical to the target TTR sequence, and 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 a nucleotide sequence equivalent region 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.

[0161] In some embodiments, an iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide, wherein the antisense polynucleotide is complementary to a target TTR sequence, and 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, 5, and 6, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2, 3, 5, and 6.

[0162] Generally, "iRNA" includes ribonucleotides with chemical modifications. Such modifications can include all types of modifications disclosed herein or known in the art. All of these modifications, when used in dsRNA molecules, are encompassed by "iRNA" for the purposes of this specification and claims.

[0163] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0164] 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.

[0165] The phrase "contacting a cell with an iRNA" such as dsRNA as used herein includes 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 performed directly or indirectly. 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 contact a cell later.

[0166] 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 are present, or by injecting iRNA into another region, for example, into the bloodstream or subcutaneous cavity, so that the agent subsequently reaches the tissue where the cells to be contacted are present.For example, iRNA can contain or be bound to a ligand, such as GalNAc, which guides iRNA to a target site, such as the liver.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.

[0167] 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.

[0168] 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.

[0169] As used herein, a "subject" is an animal, such as a mammal, including primates (such as humans, monkeys, and non-human primates such as chimpanzees), non-primates (such as cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds that express a target gene either endogenously or heterologously.

[0170] 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 expression, a human being at risk for a disease or disorder that would benefit from reduced TTR expression, a human being with a disease or disorder that would benefit from reduced TTR expression, or a human being treated for a disease or disorder that would benefit from reduced TTR 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.

[0171] In some embodiments, the human subject is suffering from a TTR-related disease. In other embodiments, the subject is at risk of developing a TTR-related disease, for example, a subject with a TTR gene mutation associated with the development of a TTR-related disease, a subject with a family history of a TTR-related disease, or a subject with signs or symptoms suggestive of the development of a TTR-related disease without meeting the diagnostic criteria for a TTR-related disease.

[0172] As used herein, the term "treat" or "treatment" refers to a beneficial or desired outcome, such as reducing at least one sign or symptom of a TTR-related disorder in a subject. Treatment also includes decreasing one or more signs or symptoms associated with unwanted TTR expression, reducing the degree of unwanted TTR activation or stabilization, or ameliorating or alleviating unwanted TTR activation or stabilization. "Treatment" can also mean increasing survival time compared to expected survival time if no treatment is administered.

[0173] The term "lower" in the context of the level of TTR or a disease marker or symptom in a subject refers to a statistically significant decrease in such level. 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. "Lowering" in the context of the level of TTR in a subject refers to a decrease to a level that is accepted as being within the normal range in individuals without the disorder. In certain embodiments, "lowering" 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 "reducing" can also be used in reference to normalizing a symptom or pathology of a disease, i.e., reducing the difference in levels between subjects with a TTR-related disorder and those with a TTR-related disorder, toward or to the level of a normal subject not afflicted with a TTR-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.

[0174] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that can be treated or ameliorated by reducing expression of the TTR gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, e.g., symptoms of TTR-related disorders such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia. A failure to develop the 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 scale clinically acceptable for the disease or disorder), or a delay in the onset of delayed symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0175] As used herein, "TTR-related disease" is intended to include any disease associated with the TTR gene or protein. Such diseases may be caused, for example, by overproduction of TTR protein, mutations in the TTR gene, abnormal cleavage of the TTR protein, instability of the TTR tetramer, or abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. "TTR-related disease" includes any type of transthyretin-mediated amyloidosis (ATTR amyloidosis) in which TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposits, such as hereditary ATTR (h-ATTR) amyloidosis or non-hereditary ATTR (ATTR) amyloidosis. TTR-related diseases include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, amyloid vitreous opacities, carpal tunnel syndrome, hyperthyroxinemia, ocular diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD) such as dry AMD and wet AMD, metabolic disorders such as impaired glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, and cardiovascular disease. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesias, hypoesthesia in the distal extremities), autonomic neuropathy (e.g., gastrointestinal disorders such as gastric ulcers or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic failure, cardiomyopathy, vitreous opacities, renal failure, nephropathy, substantial reduction in mBMI (modified body mass index), cranial nerve dysfunction, and lattice corneal dystrophy.

[0176] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a TTR-related disorder, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may 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.

[0177] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with a TTR-related disorder, is sufficient to prevent or ameliorate the disorder or one or more symptoms of the disorder. Amelioration of 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 any, and other personal characteristics of the patient to be treated.

[0178] A "therapeutically effective amount" or "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.

[0179] 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, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0180] The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle involved in the transport or transfer of the subject compound from one organ or body part to another, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, 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 harmful to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0181] 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 hepatocytes). 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.

[0182] II. iRNA of the Invention The present invention provides iRNAs that inhibit expression of the TTR gene. In certain embodiments, the iRNAs comprise double-stranded ribonucleic acid (dsRNA) molecules for inhibiting expression of the TTR gene in cells, e.g., in cells within a subject, such as a mammal, e.g., a human, predisposed to a TTR-related disorder, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia. The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed upon expression of the TTR gene. The complementary region is approximately 19-30 nucleotides in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0183] When contacted with cells expressing the TTR gene, the iRNA inhibits expression of the TTR gene (e.g., a human, primate, non-primate, or rat TTR gene) by at least about 50%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence, for example, 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 a suitable 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, for example, mice expressing a human target gene or AAV-infected mice, when administered, for example, as a single dose, at a minimum of 3 mg / kg of RNA expression.

[0184] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the 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 the mRNA formed during the expression of the TTR 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 duplex 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 as opposite each other on separate oligonucleotides.

[0185] 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 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 length. 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 intended to be part of this disclosure.

[0186] Similarly, the region of complementarity to the target sequence may be 15 to 30 nucleotides in length, e.g., 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 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.

[0187] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length.

[0188] 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 most often a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, 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).

[0189] Those skilled in the art will recognize that the duplex region is the primary functional portion of the dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. That is, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs is a dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage. Thus, those skilled 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 TTR gene expression is not generated in a target cell by cleavage of a larger dsRNA.

[0190] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. Compared with their blunt-ended counterparts, dsRNAs with at least one nucleotide overhang can have superior inhibitory properties. The nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. The overhang can be on the sense strand, the antisense strand, or any combination thereof. Moreover, the nucleotide of the overhang can be present at the 5'-end, 3'-end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0191] 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.Then, the strands of these components are 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 the oligonucleotide strands that contain 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.

[0192] 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, 5 and 6, and the corresponding antisense strand of the sense strand is selected from the sequences provided in any one of Tables 2, 3, 5 and 6.In this embodiment, one of the two sequences is complementary to the other of the two sequences, and in this case, one of the sequences is substantially complementary to the sequence of the mRNA generated during the expression of the TTR gene.Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide being described as a sense strand in any one of Tables 2, 3, 5 and 6, and the second oligonucleotide being described as the corresponding antisense strand of the sense strand in any one of Tables 2, 3, 5 and 6.

[0193] In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0194] For example, although the sequences in Table 2 are not described as modified or conjugated sequences, the RNA of the iRNA of the present invention, e.g., the dsRNA of the present invention, can comprise any one of the sequences set forth in any one of Tables 2, 3, 5, and 6, unmodified, unconjugated, or modified or conjugated differently from those set forth therein. In other words, the present invention encompasses the dsRNAs of Tables 2, 3, 5, and 6 that are unmodified, unconjugated, modified, or conjugated as described herein.

[0195] Those skilled in the art are well aware that dsRNA with a duplex structure of about 20 to 23 base pairs, for example, 21 base pairs, has been hailed as being 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 embodiment, due to the nature of the oligonucleotide sequences provided in any one of Tables 2, 3, 5 and 6, dsRNA 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, 5, and 6, minus only a few nucleotides on one or both ends, may be similarly effective compared to the dsRNAs described above. 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, 5, and 6 that differ from dsRNAs containing the entire sequence in their ability to inhibit TTR gene expression by no more than about 5, 10, 15, 20, 25, or 30% inhibition are contemplated as being within the scope of the present invention.

[0196] Furthermore, the RNAs provided in Tables 2, 3, 5, and 6 identify sites in the TTR transcript that are susceptible to RISC-mediated cleavage. Thus, the present invention also 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 consecutive nucleotides from any one of the sequences provided in any one of Tables 2, 3, 5, and 6, linked to additional nucleotide sequences taken from regions adjacent to the selected sequence in the TTR gene.

[0197] III. Modified iRNAs of the Invention In certain embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all or substantially all of the nucleotides of the 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.

[0198] 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, S.L. 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 the 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 modification or substitution of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or containing non-natural internucleoside linkages. RNAs with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes of this specification, as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. In some embodiments, modified iRNAs will have a phosphorus atom in their internucleoside backbone.

[0199] 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 agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In one embodiment, the dsRNA agent of the present invention is in sodium salt form.In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as counterion to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.The agent in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterion comprises 5, 4, 3, 2 or 1 or less phosphodiester and / or phosphorothioate linkages that do not have sodium counterion.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as counterion to all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0200] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent 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, 5,286,711, and 5,286,712. No. 7, 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. 5,47 No. 6,925, 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. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 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. 6,534, Nos. 6,639, 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 contents of each of which are incorporated herein by reference in their entirety.

[0201] Modified RNA backbones that do not contain phosphorus atoms in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain 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 constituent moieties.

[0202] Representative United States 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.

[0203] RNA mimics suitable for use in the iRNAs provided herein are contemplated, in which both the sugar and internucleoside linkages of the nucleotide units, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, in which RNA mimics have been shown to have excellent hybridization properties, is called 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 directly or indirectly linked 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. Patent 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.

[0204] 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 the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506. The natural phosphodiester backbone can be represented as OP(O)(OH)-OCH2-.

[0205] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein, may 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 10The modifications include lower 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 or 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).

[0206] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHNH), 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 a 2'-5'-linked dsRNA and the 5' position of the 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 Application Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, Nos. 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, the entire contents of each of which are incorporated herein by reference.

[0207] 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 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, J. L. ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, 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, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0208] Representative United States patents that teach the preparation of certain of the above 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.

[0209] In some embodiments, the RNAi agents of the present disclosure may 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 via a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present invention may 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 the polynucleotides of the present invention include, but are not limited to, nucleosides comprising 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 comprising a bridge from 4' to 2'.

[0210] A locked nucleoside can be represented by this structure (stereochemistry omitted): [ka]

[0211] wherein B is a nucleobase or modified nucleobase, and L is a linking group connecting the 2'-carbon and the 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). , U.S. Pat. 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.

[0212] Additional representative U.S. patents and U.S. patent application 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, 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, U.S. Patent Application Publication No. 2008 / 0039618 and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0213] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0214] 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 (i.e., 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."

[0215] 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 long enough to position the oxygen in an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0216] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0217] In some embodiments, the iRNA of the present invention includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which any of their sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also encompass 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' sugar linkage (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).

[0218] 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 Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0219] 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 WO 2011 / 005861.

[0220] Other modifications of the nucleotides of the iRNA of the present 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 Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0221] 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 agents with chemical modifications, such as those disclosed in International Publication No. 2013 / 075035, the entire contents of which are incorporated herein by reference.As shown herein and in International Publication No. 2013 / 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 completely modified otherwise.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 GalNAc derivative ligand, for example, on the sense strand.

[0222] 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.

[0223] Thus, the present invention provides double-stranded RNA agents capable of inhibiting expression of a target gene (i.e., the TTR 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.

[0224] The sense strand and antisense strand typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as a "dsRNAi agent." The duplex region of a dsRNAi agent can be, for example, a duplex 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 duplex region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0225] In certain embodiments, a dsRNAi agent may 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 may 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 may include an extended overhang region, as described above. The overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being twisted. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be linked by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0226] 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, 2'-sugar modifications, 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.

[0227] 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.

[0228] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of dsRNAi agent can be phosphorylated.In some embodiments, the overhang region contains two nucleotides with phosphorothioate between them, and these two nucleotides can be the same or different.In some embodiments, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands.In some embodiments, this 3'-overhang is present in the antisense strand.In some embodiments, this 3'-overhang is present in the sense strand.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In yet other embodiments, the dsRNAi agent is 21 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 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.

[0233] In certain embodiments, dsRNAi agent comprises a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, 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, 13 from the 5' end, and one end of the RNAi agent is blunt, and the other end comprises a 2-nucleotide overhang.In one embodiment, the 2-nucleotide overhang is at the 3' end of the antisense strand.

[0234] When a 2-nucleotide overhang is at the 3'-end of antisense strand, there can be two phosphorothioate internucleotide linkages between the three nucleotides at the end, 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 linkages between the three nucleotides at the 5'-end of sense strand and the 5'-end of antisense strand.In certain embodiments, all nucleotides in the sense strand and antisense strand of the dsRNAi agent, including the nucleotide that is part of the 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 (for example, GalNAc3).

[0235] 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'-terminal nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1 to 23 of the first strand; and the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and wherein the 5'-end of the antisense strand comprises 10 to 30 consecutive ribonucleotides that are not paired with the sense strand. the sense strand comprises nucleotides following the nucleotide sequence of the antisense 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 the double-stranded nucleic acid reduces target gene expression when introduced into a mammalian cell, and the sense strand contains 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 contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0236] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, the dsRNAi agent comprises 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 the 11th, 12th, and 13th positions from the 5'-end, the 3'-end of the first strand and the 5'-end of the second strand form a blunt end, and the second strand is 1-4 nucleotides longer than the first strand at its 3'-end, the duplex region is at least 25 nucleotides long, and the second strand is sufficiently complementary to target mRNA along at least 19 nucleotides of the length of the second strand, and when the RNAi agent is introduced into mammalian cells, it reduces target gene expression, and Dicer cleavage of the dsRNAi agent results in siRNA comprising the 3'-end of the second strand, thereby reducing the expression of target gene in mammalian cells.Optionally, the dsRNAi agent further comprises a ligand.

[0237] 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.

[0238] 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.

[0239] For dsRNAi agents having a duplex 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 these numbers start from the first nucleotide from the 5' end of the antisense strand, or these numbers start from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the dsRNAi agent from the 5' end.

[0240] The sense strand of dsRNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the break site of strand, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the break site 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.

[0241] 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 motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif that occurs in another part of the strand that is separated from a motif at or near the cleavage site of the same strand. The wing modification is adjacent to the first motif or is 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.

[0242] Similar to sense strand, the antisense strand of dsRNAi agent can contain two or more motifs of three identical modifications on three consecutive nucleotides, and at least one of these motifs occurs at or near the break site of strand.This antisense strand can also contain one or more wing modifications in the same alignment as the wing modifications that can exist on sense strand.

[0243] 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.

[0244] 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 duplex region at the 3' end, 5' end, or both ends of the strand.

[0245] When the sense and antisense strands of a dsRNAi agent each contain at least one wing modification, the wing modifications can be at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0246] 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 duplex region with an overlap of 1, 2, or 3 nucleotides, two modifications from each single strand are at the other end of a duplex 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 duplex region with an overlap of 1, 2, or 3 nucleotides.

[0247] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNAi agent, including the nucleotide that is part of motif, can be modified.Each nucleotide can be modified with the same or different modifications, and this modification can include one or more of the following: one or both of non-linked phosphate oxygen or one or more of linked phosphate oxygens, the modification of ribose sugar components, for example, the modification of the 2' hydroxyl on ribose sugar, the large-scale replacement of phosphate moiety with " dephosphorylation " linker, the modification or replacement of naturally occurring base, and the replacement or modification of ribose phosphate backbone.

[0248] Because nucleic acid is a polymer of subunits, many modifications occur at positions that are repeated within nucleic acid, such as modifications of bases or phosphate moieties, or unlinked O of phosphate moieties.In some cases, modifications occur at all target positions in nucleic acid, but in many cases, they do not occur.For example, modifications can occur only at the 3'-end or 5'-end position, or only at terminal regions, such as at the terminal nucleotide position of the chain, or at 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 in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both termini, can occur only in terminal regions, e.g., at the terminal nucleotide of the strand or within the last 2, 3, 4, 5, or 10 nucleotides of the strand, or can occur in double-stranded and single-stranded regions, especially at the termini. The 5' termini can be phosphorylated.

[0249] This may, for example, enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' overhang 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 part of the bases in the 3' overhang or 5' overhang can be modified, for example, with the modifications described herein.Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, for example, the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, instead of the ribosugar of the nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0250] In some embodiments, each residue of sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.Strands can contain two or more modifications.In one embodiment, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0251] At least two different modifications are typically present on the sense and antisense strands, which may be 2'-O-methyl or 2'-fluoro modifications, for example.

[0252] In certain embodiments, N a or N b includes alternating patterns of modifications. The term "alternating motif," as used herein, refers to a motif having one or more modifications, each modification occurring on alternating nucleotides in a single strand. Alternating nucleotides can refer to one every other nucleotide or one every three nucleotides, or similar patterns. 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.

[0253] 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 within the alternating motif, such as "ABABAB...," "ACACAC...," "BDBDBD...," or "CDCDCD...."

[0254] In some embodiments, the dsRNAi agents of the present invention comprise an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. This shift can be made so that the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides 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 duplex 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 and antisense strands.

[0255] In some embodiments, dsRNAi agent comprises the alternating motif pattern of 2'-O-methyl modification and 2'-F modification on sense strand, and first has a relative shift with respect to the alternating motif pattern 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.

[0256] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into sense strand or antisense strand interrupts the original modification pattern existing in sense strand or antisense strand.The interruption of the modification pattern of sense strand or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into sense strand or antisense strand can enhance the gene silencing activity for target gene.

[0257] In some embodiments, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotide adjacent to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif may be designated "...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.

[0258] 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 along the strand. For example, the internucleotide linkage modification may occur at any nucleotide on the sense strand or the antisense strand, and 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 relative shift 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.

[0259] In some embodiments, dsRNAi agent comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region can contain two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between two nucleotides.Internucleotide linkage modification can also be made to connect overhang nucleotide with the terminal paired nucleotide in double-stranded region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and optionally there can be another phosphorothioate or methylphosphonate internucleotide linkage that connects overhang nucleotide with the paired nucleotide adjacent to overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to 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.

[0260] In some embodiments, there is a 2-nucleotide overhang at the 3'-end of 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 also have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of sense strand and the 5'-end of antisense strand.

[0261] In one embodiment, the dsRNAi agent comprises mismatches or combinations thereof in the duplex with the target. Mismatches can occur in the overhang region or in the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but then adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, for example, non-standard pairings or non-standard pairings (described elsewhere in this specification), are more preferable than standard (A:T, A:U, G:C) pairings, and pairings that include universal bases are more preferable than standard pairings.

[0262] In certain embodiments, the dsRNAi agent comprises the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and at least one mismatch pair, e.g., a non-canonical pairing or a non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0263] 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 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0264] 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, such as two dT nucleotides on the 3' end of the sense strand, the antisense strand, or both strands.

[0265] 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 each independently represent 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 represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides, 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. In some embodiments, N a or N b includes alternating pattern modifications.

[0266] In some embodiments, YYY motif occurs at or near the cleavage site of sense strand.For example, when dsRNAi agent has the double-stranded region of 17-23 nucleotides in length, YYY motif can occur at or near the cleavage site of sense strand (for example, can occur at 6,7,8 position, 7,8,9 position, 8,9,10 position, 9,10,11 position, 10,11,12 position, or 11,12,13 position), and this number starts from the first nucleotide from 5' end, or optionally this number starts from the first paired nucleotide in double-stranded region from 5' end.

[0267] 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 following 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).

[0268] 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 containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0269] 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 containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0270] 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.

[0271] Each of X, Y and Z may be the same or different from one another.

[0272] 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). When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0273] 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 each independently represent 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 represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represent 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. In some embodiments, N a ' or N b ' includes alternating pattern modifications.

[0274] Y'Y'Y' motif occurs at or near the cleavage site of antisense strand.For example, when dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, Y'Y'Y' motif can occur at the 9th, 10th, 11th, 10th, 11th, 12th, 11th, 12th, 13th, 12th, 13th, 14th, or 13th, 14th, 15th positions of antisense strand, and this number starts from the first nucleotide from the 5' end, or optionally this number starts from the first paired nucleotide in double-stranded region from the 5' end.In one embodiment, Y'Y'Y' motif occurs at the 11th, 12th, 13th positions.

[0275] In certain embodiments, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0276] 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.

[0277] Thus, the antisense strand has the 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).

[0278] 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 represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0279] 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 represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0280] When the antisense strand is represented by formula (IId), each 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. aIn 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.

[0281] 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).

[0282] When the antisense strand is represented by formula (IIa), each N a ' independently represent 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.

[0283] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0284] In some embodiments, the sense strand of the dsRNAi agent can contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21nt (nucleotides), the numbers starting from the first nucleotide from the 5' end, or optionally the numbers starting from the first paired nucleotide in the duplex 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 duplex region, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0285] In some embodiments, the antisense strand may 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 duplex region from the 5' end, and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0286] 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).

[0287] 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 each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; In the formula, each n p ',n p , n q ' and n q each of which may be present or absent independently represents an overhanging nucleotide; 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.

[0288] 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.

[0289] 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'-Nb ’ -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).

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from one another.

[0295] 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.

[0296] When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can form a base pair with one of the Z' nucleotides. Alternatively, at least two Z nucleotides form a base pair with the corresponding Z' nucleotide, or all three Z nucleotides form a base pair with the corresponding Z' nucleotide.

[0297] When the dsRNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can form a base pair with one of the X' nucleotides.Alternatively, at least two of the X nucleotides can form a base pair with the corresponding X' nucleotide, or all three of the X nucleotides can form a base pair with the corresponding X' nucleotide.

[0298] 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.

[0299] 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 other embodiments, 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 is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker (described below). a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0300] In some embodiments, when the dsRNAi agent has Formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0301] In some embodiments, dsRNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and said double strands are connected by linker.This linker can be cleavable or not cleavable.Optionally, said multimer further comprises ligand.Each double strand can target the same gene or two different genes, or each double strand can target the same gene at two different target sites.

[0302] In some embodiments, dsRNAi agent is a multimer that contains three, four, five, six or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and said double strands are connected by linker.This linker can be cleavable or not cleavable.Optionally, said multimer further comprises a ligand.Each double strand can target the same gene or two different genes, or each double strand can target the same gene at two different target sites.

[0303] 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.

[0304] 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.

[0305] In other embodiments, the RNAi agent of the present invention may contain a very small number of nucleotides containing 2'-fluoro modifications, for example, two or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain two, one, or zero nucleotides with 2'-fluoro modifications. In certain embodiments, the 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.

[0306] Various publications describe multimeric iRNAs that can be used in the methods of the invention, including WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0307] 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;

[0308] R is hydrogen, hydroxy, fluoro, or C 1~20 alkoxy (e.g., methoxy or n-hexadecyloxy);

[0309] R 5’ is =C(H)-P(O)(OH)2, and the C5' carbon and R 5’ and the double bond between

[0310] B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0311] The vinyl phosphonate 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 phosphonate of the present disclosure is attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0312] Vinyl phosphonate modifications are also contemplated in the compositions and methods of the present disclosure. Exemplary vinyl phosphonate structures include those described above, where R is =C(H)-O-P(O)(OH) and the double bond between the C carbon and R is in the E or Z configuration (e.g., the E configuration).

[0313] 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 carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be heteroatoms, 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.

[0314] 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 "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally refers 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 "tethering attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (other than the atom that provides the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., 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 often contain functional groups, e.g., amino groups, or generally bonds, that provide a linkage suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, to the constituent ring.

[0315] The iRNA can be conjugated to the ligand via a carrier, and the carrier can be a cyclic group or an 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.

[0316] i. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermally destabilizing 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, thermally destabilizing modifications can be incorporated into the seed region of antisense strand to reduce or inhibit off-target gene silencing.

[0317] 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.

[0318] 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., one, two, three, four, five, 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, one or more thermally destabilizing modifications of the duplex are located at positions 2-9, for example, 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 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.

[0319] An iRNA agent includes a sense strand and an antisense strand, each strand having 14-40 nucleotides. The RNAi agent has the following formula (L): [ka] (L), which can be expressed as: 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.

[0320] C1 is a thermally destabilizing 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 thermally destabilizing 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 in a position that: i) mismatches 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] where B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar modification. In one embodiment, the thermally destabilizing 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 thermally destabilizing modification in C1 is GNA or [ka] is. T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides the nucleotide with steric bulk equal to or less than that of 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 may be at the 2'-position of the ribose sugar of the nucleotide, or may 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 that 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. n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length. n 5, q 3 , and q 7 are independently 1 to 6 nucleotides in length. n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length, or n 4 is 0 nucleotides long. q 5 are independently 0 to 10 nucleotides in length. n 2 , and q 4 are independently 0 to 3 nucleotides in length.

[0321] Or, n 4 is 0 to 3 nucleotides in length.

[0322] In one embodiment, n 4 can be 0. In one embodiment, n 4 is 0 and q 2 and q 6 is 1. In another embodiment, n 4 is 0 and q 2 and q 6 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 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand).

[0323] In one embodiment, n 4 , q 2 , and q 6 are each 1.

[0324] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each 1.

[0325] 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.

[0326] 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.

[0327] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. 2 is equal to 1.

[0328] 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.

[0329] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0330] In one embodiment, T1' is at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand, and 2 is equal to 1, and non-ribose acyclic or backbone modifications at the 2' position are less sterically bulky than 2'-OMe ribose.

[0331] 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, and6 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.

[0332] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is 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 2 When 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.

[0333] 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.

[0334] 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 intrabackbone position that is less sterically bulky than 2'-OMe ribose.

[0335] In one embodiment, T2' starts at position 8 from the 5' end of the antisense strand. In one example, T2' starts at position 8 from the 5' end of the antisense strand and 4 is 2.

[0336] In one embodiment, T2' begins at position 9 from the 5' end of the antisense strand. 4 is 1.

[0337] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, 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).

[0338] 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 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).

[0339] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and 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.

[0340] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 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'-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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0341] 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.

[0342] 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 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, 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).

[0343] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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.

[0344] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 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 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0345] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 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 2 additional TTs at the 3' end of the antisense strand.

[0346] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, optionally with at least two additional TTs at the 3' end of the antisense strand, 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 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0347] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0348] 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 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 of the antisense strand (counting from the 5' end).

[0349] 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'-F, and q 7 is 1.

[0350] 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 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).

[0351] 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'-F, and q 7 is 1.

[0352] 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 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).

[0353] 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., a trans-vinyl phosphonate, [ka] ), 5'-Z-VP isomer (i.e., cis-vinyl phosphonate, [ka] ) or a mixture thereof. 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.

[0354] In one embodiment, the RNAi agent comprises a 5'-P. In one embodiment, the RNAi agent comprises a 5'-P in the antisense strand.

[0355] In one embodiment, the RNAi agent comprises a 5'-PS. In one embodiment, the RNAi agent comprises a 5'-PS in the antisense strand.

[0356] 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.

[0357] In one embodiment, the RNAi agent comprises a 5'-PS2. In one embodiment, the RNAi agent comprises a 5'-PS2 in the antisense strand.

[0358] 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. 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'-PS.

[0359] 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'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0360] 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'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0361] 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'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-PS2.

[0362] 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. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0363] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6is 1, B4' is 2'-OMe, and q 7 is 1, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-P.

[0364] 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-PS.

[0365] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, 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 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. 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 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-PS2.

[0366] 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0367] 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'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0368] 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.

[0369] 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. 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. 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.

[0370] 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'-OMe, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0371] 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 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 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-P.

[0372] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 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 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS.

[0373] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 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 of the antisense strand (counting from the 5' end). 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.

[0374] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 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 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS2.

[0375] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and 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'-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 of the antisense strand (counting from the 5' end). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0376] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0377] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0378] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 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. In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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 dsRNAi RNA agent also includes a 5'-PS2.

[0379] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0380] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-P.

[0381] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-PS.

[0382] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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 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.

[0383] 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 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-PS2.

[0384] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0385] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 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.

[0386] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0387] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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.

[0388] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q5 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.

[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 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.

[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'-F, and q7 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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-P.

[0391] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-PS.

[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 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 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.

[0393] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 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 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-PS2.

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

[0395] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 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, 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 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.

[0396] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and 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 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.

[0397] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-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 (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof), and a targeting ligand.

[0398] 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.

[0399] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-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 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.

[0400] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1, 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 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.

[0401] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 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 of the antisense strand (counting from the 5' end). 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.

[0402] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0403] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-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 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In 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.

[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 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'-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 of the antisense strand (counting from the 5' end). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[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 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 of the antisense strand (counting from the 5' end). 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.

[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'-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 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.

[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 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 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.

[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 q3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1, 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 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.

[0409] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, 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 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.

[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'-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 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.

[0411] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7 and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 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 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.

[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 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 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.

[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 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 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.

[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'-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 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.

[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'-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 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.

[0416] 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.

[0417] 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) 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.

[0418] 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) 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.

[0419] 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) 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.

[0420] 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) 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.

[0421] 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) 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.

[0422] 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) 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.

[0423] 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) 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.

[0424] 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) 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.

[0425] 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) 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.

[0426] In certain embodiments, the iRNA used in the methods of the invention is an agent selected from the agents listed in any one of Tables 2, 3, 5, and 6. These agents may further comprise a ligand.

[0427] 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. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556).In other embodiments, the ligand is cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether 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), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain such as a dodecanediol or undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118, Kabanov et al., FEBS Lett., 1990, 259:327-330, Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, such as 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).

[0428] In certain embodiments, the ligand changes the distribution, targeting, or life span of the iRNA agent into which it is incorporated.In some embodiments, the ligand enhances the affinity of a selected target, for example, a molecule, a cell or cell type, a compartment such as a cell or organ compartment, tissue, organ or region of the body, for example, when compared with a species in which such a ligand is not present.In some embodiments, the ligand does not participate in double-stranded pairing in double-stranded nucleic acid.

[0429] 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.

[0430] The ligand can also include a targeting group, such as a cell or tissue targeting agent, for example, a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as 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 acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimic. In certain embodiments, the ligand is a polyvalent galactose, such as N-acetyl-galactosamine.

[0431] 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)lithophosphate, and the like. Examples of suitable cleavage inhibitors include oleic 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.

[0432] A ligand can be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, such as a liver cell. 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.

[0433] The ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0434] In some embodiments, the ligands binding 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 have also been shown to bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0435] The ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing reactive functional pendant side chains, such as those derived 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.

[0436] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely produced by the well-known technique of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, California).Any other method for such synthesis known in the art can additionally or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0437] 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 nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or ligand-nucleoside conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0438] When using a nucleotide-conjugate precursor that already possesses a linking moiety, typically, synthesis of the sequence-specifically linked nucleoside is 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.

[0439] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule.In one embodiment, this lipid or lipid-based molecule binds serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue in the body, for example, to target tissue other than the kidney.For example, the target tissue can be the liver, including the parenchymal cells of the liver.Other molecules that can bind HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) can be used to adjust the binding to serum protein, for example, HSA.

[0440] 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 kidney, and therefore is less likely to be eliminated from body.The lipid or lipid-based ligand that binds less strongly to HSA can be used so that conjugate targets kidney.

[0441] 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.

[0442] 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.

[0443] In another embodiment, the ligand is a moiety, for example, a vitamin, that is taken up by target cells, for example, proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells.Exemplary vitamins include vitamins A, E, and K.Other exemplary vitamins include vitamin B, for example, folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, such as hepatocytes.Also included are HSA and low-density lipoprotein (LDL).

[0444] 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 antennapedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidyl mimics, 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.

[0445] 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 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0446] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can contain a hydrophobic membrane translocating sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). RFGF analogs containing a 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, such as from phage display libraries or one-bead-one compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 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 affect conformational properties. Any of the structural modifications described below can be utilized.

[0447] The RGD peptide 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 peptidomimetics can contain D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands, such as PECAM-1 or VEGF.

[0448] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, 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).

[0449] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for 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 its part 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. Representative 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).

[0450] In certain embodiments, the carbohydrate conjugates used in the compositions and methods of the present invention are monosaccharides.

[0451] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in U.S. Patent 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).

[0452] 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 the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., via a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., via a linker as described herein.

[0453] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a tetravalent linker.

[0454] In certain embodiments, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative linked to an iRNA agent. In certain embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0455] In some embodiments, for example, when the two strands of an iRNA agent of the present invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the corresponding other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently contain a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0456] In some embodiments, for example, when the two strands of an iRNA agent of the present invention are part of a larger molecule connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the corresponding other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently contain a GalNAc or GalNAc derivative linked via a monovalent linker. This hairpin loop can also be formed by an extended overhang in one strand of the duplex.

[0457] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: [ka] Formula II, [ka] Formula III, [ka] Formula IV, [ka] Formula V, [ka] Formula VI,

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[0458] In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine, e.g. [ka] Formula II, etc.

[0459] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S: [ka]

[0460] In some embodiments, the RNAi agent is conjugated to L96, as defined in Table 1 and shown below: [ka]

[0461] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] (Formula XXXVI), and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0462] In some embodiments, suitable ligands are those disclosed in WO 2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand comprises the following structure: [ka]

[0463] In certain embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker.

[0464] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives linked to the iRNA agent. GalNAc can be linked to any nucleotide via a linker on the sense strand or antisense strand. GalNAc can be linked to the 5'-end of the sense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, or the 3'-end of the antisense strand. In one embodiment, GalNAc is linked to the 3'-end of the sense strand, for example, via a trivalent linker.

[0465] In other embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple linkers, e.g., monovalent linkers.

[0466] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0467] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0468] Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in PCT Publication Nos. WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0469] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which may or may not be cleavable.

[0470] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently bonds the two parts of a compound.Typically, a linker is a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkynyl, alkenylarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl and the like, wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle, where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18, 7 to 17, 8 to 17, 6 to 16, 7 to 17, or 8 to 16 atoms.

[0471] A cleavable linking group is one that is sufficiently stable outside a cell, but that, once inside a target cell, is cleaved to release the two moieties held together by the linker. In an exemplary embodiment, the cleavable linking group is cleaved at a rate that is at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least 100-fold faster in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0472] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Cleavage agents are generally more widespread or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a specific substrate or have no substrate specificity, e.g., oxidases or reductases or reducing agents present in cells, such as mercaptans, that can degrade redox-cleavable linking groups by reduction; esterases; reagents that can create an endosomal or acidic environment, e.g., reagents that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0473] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a selected pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0474] Linker can comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the target cell.For example, liver targeting ligand can be linked to cationic lipid via a linker that comprises an ester group.Hepatocytes are rich in esterase, so linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0475] When targeting cell types rich in peptidases, such as hepatocytes and synoviocytes, linkers containing peptide bonds can be used.

[0476] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, where the first condition is selected to exhibit cleavage in target cells, and the second condition is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In certain embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0477] i. Redox-cleavable linking group In certain embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. For example, candidates can be evaluated in cells by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some conditions, the candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at a rate at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0478] ii. Phosphate-based cleavable linkers In other embodiments, the cleavable linker comprises a phosphate-based cleavable linking group that is cleaved by a reagent that degrades or hydrolyzes the phosphate group. An example of a reagent that cleaves the phosphate group in a cell is an enzyme such as a phosphatase in the cell. Examples of phosphate-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-, where Rk in each occurrence can independently be C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In certain embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0479] iii. Acid-cleavable linking group In other embodiments, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In certain embodiments, the acid-cleavable linking group is cleaved in an acidic environment where the pH is about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower) or by a reagent, such as an enzyme, that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). Exemplary embodiments include, when the carbon is bonded to the oxygen of the ester (alkoxy group), an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0480] iv. Ester-based linking group

[0481] In other embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0482] v. Peptide-based cleavage groups

[0483] In yet other embodiments, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved in cells by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to yield peptides and proteins, and do not include all amide functional groups. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0484] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to: [ka] (formula XXXVII), [ka] (formula XXXVIII), [ka] (formula XXXIX), [ka] (formula XL), [ka] (formula XLI), [ka] (formula XLII), [ka] (Formula XLIII), and [ka] (Formula XLIV), and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0485] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0486] In one embodiment, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of the following formulas (XLV) to (XLVIII): [ka] During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5Cis, independently at each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O), R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C are each independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represents a ligand, i.e., independently at each occurrence a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide, and R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for use with RNAi agents that inhibit expression of target genes, such as those of formula (XLIX): formula XLIX [ka] In the formula, L 5A , L 5B , and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0487] Examples of bivalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above, such as Formulas II, VII, XI, X, and XIII.

[0488] Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, and 5,414,078. No. 7, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4,605,735, No. 4,667,025, No. 4,762,779, No. 4,789,73 No. 7, No. 4,824,941, No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,082,830 No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098 No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810 Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0489] Not all positions in a given compound need be uniformly modified, and in fact more than one of the foregoing modifications can be incorporated within a single compound, or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0490] A "chimeric" iRNA compound or "chimera," in the context of this invention, is an iRNA compound, such as a dsRNAi agent, that contains two or more chemically distinct regions, each of which is composed of at least one monomeric unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNA when using chimeric dsRNA compared to the phosphorothioate deoxydsRNA hybridized to the same target region.Cleavage of RNA target can be routinely detected by gel electrophoresis, and if necessary, by related nucleic acid hybridization techniques known in the art.

[0491] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to enhance their activity, cellular distribution, or cellular uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using an appropriate coupling or activation reagent. The conjugation reaction can be performed while the RNA is still attached to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0492] IV. Delivery of iRNA of the Invention Delivery of iRNAs of the invention to cells, e.g., in a subject in need thereof, such as a human subject (e.g., a subject susceptible to or diagnosed with a TTR-related disorder, e.g., a subject suffering from a TTR-related disorder, e.g., senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, ocular diseases, e.g., Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), e.g., dry AMD and wet AMD, metabolic disorders, e.g., disorders of glucose and lipid homeostasis, e.g., insulin resistance associated with type II diabetes, cardiovascular disease), can be achieved in many different ways. For example, delivery can be performed by contacting cells with iRNAs of the invention either in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition containing an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that encode and induce the expression of the iRNA. These options are further described below.

[0493] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider for delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. RNA interference has also been shown to be successful in localized delivery to the CNS via direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602). Modification of RNA or pharmaceutical carriers can also enable targeting of iRNA to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, iRNAs directed against ApoB conjugated to lipophilic cholesterol moieties were systemically injected into mice, resulting in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).

[0494] In alternative embodiments, iRNAs can be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote binding of iRNA molecules (which are negatively charged) and also enhance their interaction with the negatively charged cell membrane, thereby enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can bind to iRNAs or can be induced to form vesicles or micelles that encapsulate iRNAs (see, e.g., Kim SH, et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles also prevents degradation of iRNAs when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, DR, et al (2003) J. Mol. Biol 327:761-766; Verma, UN, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN, et al (2003), supra), "solid nucleic acid lipid particles" (Zimmermann, TS, et al (2006) Nature 441:111-114), cardiolipin (Chien, PY, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, DA, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, the iRNA is complexed with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of iRNA and cyclodextrin can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety. A particular aspect of the present disclosure relates to a method of reducing expression of the TTR gene in a cell, comprising contacting the cell with a double-stranded RNAi agent of the present disclosure. In one embodiment, the cell is a hepatic cell, optionally a hepatocyte. In one embodiment, the cell is an extrahepatic cell.

[0495] Vectors encoding the iRNAs of the present invention iRNAs targeting the TTR gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al., PCT International Application Publication No. WO 00 / 22113; Conrad, PCT International Application Publication No. WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (on the order of hours to weeks) or persistent (weeks to months or longer), depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative. The transgene can also be constructed to allow it to be propagated as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0496] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors, (b) retrovirus vectors, such as, but not limited to, lentivirus vectors and Moloney murine leukemia virus, (c) adeno-associated virus vectors, (d) herpes simplex virus vectors, (e) SV40 vectors, (f) polyomavirus vectors, (g) papillomavirus vectors, (h) picornavirus vectors, (i) poxvirus vectors, such as orthopox, such as vaccinia virus vectors, or avian pox, such as canarypox or fowlpox, and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not become integrated into the cellular genome. The construct can include viral sequences for transfection, if desired. Alternatively, the constructs can be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNAs will generally require regulatory elements, such as promoters, enhancers, etc., to ensure expression of the iRNA in target cells. Other aspects of vector and construct considerations are known in the art.

[0497] V. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations containing the iRNA of the present invention. In one embodiment, a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier is provided herein. The pharmaceutical composition containing the iRNA is useful for preventing or treating TTR-related disorders, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia.

[0498] Such pharmaceutical compositions are formulated based on the mode of delivery. An example is a composition formulated for systemic administration by parenteral delivery, such as subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the present invention can be administered in a dosage sufficient to inhibit the expression of the TTR gene.

[0499] In some embodiments, the pharmaceutical compositions of the present invention are sterile, hi other embodiments, the pharmaceutical compositions of the present invention are pyrogen-free.

[0500] The pharmaceutical compositions of the present invention can be administered at a dosage sufficient to inhibit TTR gene expression. Generally, suitable doses of iRNAs of the present invention will range from about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally from about 1 to 50 mg per kilogram of recipient body weight per day. Typically, suitable doses of iRNAs of the present invention will range from about 0.1 mg / kg to about 5.0 mg / kg, e.g., from about 0.3 mg / kg to about 3.0 mg / kg. Repeated administration regimens can include periodic administration of a therapeutic amount of iRNA, e.g., monthly, every 3 to 6 months, or once a year. In certain embodiments, iRNAs are administered approximately once a month to approximately once every 6 months.

[0501] After the initial treatment regimen, treatment can be administered less frequently. The duration of treatment can be determined based on the severity of the disease.

[0502] In other embodiments, a single dose of the pharmaceutical composition may be administered over a long period of time, with the doses administered at intervals of one month or less, two months or less, three months or less, or four months or less. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered approximately once a month. In other embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered four times a year (i.e., approximately once every three months). In other embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered twice a year (i.e., approximately once every six months).

[0503] Those skilled in the art will understand that certain factors, such as, but not limited to, mutations present in the subject, previous treatments, the subject's general health or age, and other diseases present, can affect the dosage and timing of dosages required to effectively treat a subject. Moreover, treatment of a subject with a prophylactically or therapeutically effective amount of a composition can include a single treatment or a series of treatments, as appropriate.

[0504] The pharmaceutical composition of the present disclosure can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated.Administration can be topical (for example, intraocular, vaginal, rectal, intranasal, transdermal, etc.), oral, or parenteral administration.Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, for example, subcutaneous administration by an implanted device, or intracranial administration, for example, by intraparenchymal, intrathecal, or intraventricular administration.

[0505] The iRNA can be delivered in a manner that targets a specific tissue, such as the liver.

[0506] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the RNAi agent featured in this disclosure is mixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), cationic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The RNAi agents featured in this disclosure can be encapsulated in liposomes or complexed with liposomes, particularly cationic liposomes. Alternatively, the RNAi agents can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monoolein, dilaurin, 1-glyceryl monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C1-20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0507] In one embodiment, the double-stranded RNA agent, siRNA, of the present invention is administered to cells as a pharmaceutical composition by a local administration route. In one embodiment, the pharmaceutical composition can include the siRNA compound mixed with a topical delivery agent. The topical delivery agent can be a plurality of microvesicles. The microvesicles can be liposomes. In some embodiments, the liposomes are cationic liposomes.

[0508] In another embodiment, dsRNA agent is mixed with topical penetration enhancer.In one embodiment, topical penetration enhancer is fatty acid.Fatty acid can be arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monoolein, dilaurin, 1-glyceryl monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C1-10 alkyl ester, monoglyceride, diglyceride, or its pharmaceutically acceptable salt.

[0509] In another embodiment, the topical penetration enhancer is a bile salt, which can be cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether, or a pharmaceutically acceptable salt thereof.

[0510] In another embodiment, the topical penetration enhancer is a chelating agent, which may be EDTA, citric acid, salicylate, N-acyl derivatives ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a salt thereof for inhibiting the expression of transthyretin (TTR) in a cell, comprising: the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region; the nucleotide sequence of the sense strand differs by no more than four bases from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' of SEQ ID NO:21, and the nucleotide sequence of the antisense strand differs by no more than four bases from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3' of SEQ ID NO:22; wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; and A double-stranded ribonucleic acid (dsRNA) agent or salt thereof, wherein at least one strand is conjugated to a ligand.

2. (a) the nucleotide sequence of the sense strand differs by no more than three bases from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' of SEQ ID NO:21, and the nucleotide sequence of the antisense strand differs by no more than three bases from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3' of SEQ ID NO:22; (b) the nucleotide sequence of the sense strand differs by no more than two bases from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' of SEQ ID NO:21, and the nucleotide sequence of the antisense strand differs by no more than two bases from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3' of SEQ ID NO:22; (c) the nucleotide sequence of the sense strand differs by no more than one base from the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuuacu-3' of SEQ ID NO:21, and the nucleotide sequence of the antisense strand differs by no more than one base from the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3' of SEQ ID NO:22; (d) the sense strand comprises the nucleotide sequence of SEQ ID NO: 21, 5'-csasagagUfaUfUfCfcauuuuuuacu-3', and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 22, 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3'; and / or (e) the sense strand consists of the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuuacu-3' of SEQ ID NO: 21, and the antisense strand consists of the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3' of SEQ ID NO: 22; 2. The dsRNA agent of claim 1, or a salt thereof.

3. 3. The dsRNA agent, or salt thereof, of claim 1 or 2, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

4. 3. The dsRNA agent, or salt thereof, of claim 1 or 2, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

5. 3. The dsRNA agent or salt thereof of claim 1 or 2, wherein said ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

6. 5. The dsRNA agent of claim 4, wherein the ligand is: 【Chemical 1】

7. wherein the dsRNA agent is conjugated to the ligand as shown in the diagram below: 【Chemistry 2】 7. The dsRNA agent of claim 6, wherein X is O or S.

8. 8. The dsRNA agent of claim 7, wherein said X is O.

9. The method of claim 1, wherein the sense strand comprises the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' of SEQ ID NO: 21, and the antisense strand comprises the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3' of SEQ ID NO: 22; wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; and The dsRNA agent is conjugated to a ligand as shown in the diagram below. 【Chemistry 3】 wherein X is O; 3. The dsRNA agent or salt thereof of claim 1 or 2.

10. The sense strand comprises the nucleotide sequence 5'-csasagagUfaUfUfCfcauuuuuacu-3' of SEQ ID NO: 21, and the antisense strand comprises the nucleotide sequence 5'-asGfsuaaAfaauggaaUfaCfucuugsgsu-3' of SEQ ID NO: 22, wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; and The dsRNA agent is conjugated to a ligand as shown in the diagram below. 【Chemistry 4】 wherein X is O; 10. The dsRNA agent of claim 9, or a salt thereof.

11. 10. An in vitro cell containing the dsRNA agent of claim 1 or 2, or a salt thereof.

12. 10. A pharmaceutical composition for inhibiting expression of a gene encoding transthyretin (TTR), comprising the dsRNA agent of claim 1 or 2, or a salt thereof, and a pharmaceutically acceptable carrier.

13. 13. The pharmaceutical composition of claim 12, wherein the dsRNA agent or salt thereof is in an unbuffered solution.

14. 14. The pharmaceutical composition of claim 13, wherein the non-buffered solution is saline or water.

15. 13. The pharmaceutical composition of claim 12, wherein the dsRNA agent or salt thereof is in a buffered solution.

16. 16. The pharmaceutical composition of claim 15, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

17. 16. The pharmaceutical composition of claim 15, wherein the buffer solution is phosphate buffered saline (PBS).

18. 10. An in vitro method for inhibiting expression of the transthyretin (TTR) gene in a cell, comprising contacting the cell with a dsRNA agent of claim 1 or 2, or a salt thereof, or a pharmaceutical composition for inhibiting expression of a gene encoding transthyretin (TTR), comprising the dsRNA agent of claim 1 or 2, or a salt thereof, thereby inhibiting expression of the TTR gene in the cell.

19. 10. A pharmaceutical composition for treating a subject suffering from or at risk of developing a TTR-related disease, comprising a therapeutically or prophylactically effective amount of the double-stranded RNAi agent of claim 1 or 2, or a salt thereof.

20. (a) The subject is a human; (b) the subject is suffering from a TTR-related disease; (c) the subject is at risk of developing a TTR-associated disease; (d) the subject carries a TTR gene mutation associated with the development of a TTR-related disease; or (e) the subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the composition reduces amyloid TTR deposits in the subject, and optionally the ATTR is hereditary ATTR (h-ATTR), or The ATTR is a non-inherited ATTR (wt ATTR).

20. The pharmaceutical composition of claim 19.

21. (a) The TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia; (b) the TTR-related disease is an ocular disease; optionally, the ocular disease is Stargardt's disease, diabetic retinopathy, or age-related macular degeneration (AMD); (c) the TTR-related disease is a metabolic disorder; optionally, the metabolic disorder is a disorder of glucose and lipid homeostasis, and further optionally, the disorder of glucose and lipid homeostasis is insulin resistance associated with type II diabetes; or (d) the TTR-related disease is a cardiovascular disease; 20. The pharmaceutical composition of claim 19.

22. 20. The pharmaceutical composition of claim 19, wherein administration of the pharmaceutical composition to the subject improves at least one indicator of neuropathy, quality of life, ongoing neurological damage, or cardiovascular disorder in the subject.

23. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is administered by subcutaneous or intravenous administration.

24. 24. The pharmaceutical composition of claim 23, wherein the subcutaneous administration is self-administration.

25. 25. The pharmaceutical composition of claim 24, wherein the self-administration is via a pre-filled syringe or an auto-injector syringe.

26. 20. The pharmaceutical composition of claim 19, further comprising assessing the level of TTR mRNA or TTR protein expression in a sample from the human subject.

27. 10. A kit, vial, or syringe comprising the dsRNA agent of claim 1 or 2, or a salt thereof, or a pharmaceutical composition for inhibiting expression of a gene encoding transthyretin (TTR), comprising the dsRNA agent of claim 1 or 2, or a salt thereof.

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