TRANSTHYRETIN (TTR) iRNA COMPOSITIONS AND METHODS OF USE THEREOF FOR TREATING OR PREVENTING TTR-ASSOCIATED DISEASES

Abstract

To provide an effective therapeutic method for TTR-associated diseases.SOLUTION: The present invention provides iRNA agents, for example, double-stranded iRNA agents, that target the transthyretin (TTR) gene, and methods of using such iRNA agents for treating or preventing TTR-associated diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 199,563, filed July 31, 2015, and U.S. Provisional Patent Application No. 62 / 287,518, filed January 27, 2016. The entire contents of each of the foregoing applications are hereby incorporated by reference herein.

[0002] This application is related to U.S. Provisional Patent Application No. 61 / 881,257, filed September 23, 2013, and International Application No. PCT / US2014 / 056923, filed September 23, 2014, the entire contents of each of which are hereby incorporated by reference. Additionally, this application is related to U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, International Application No. PCT / US2012 / 065601, filed November 16, 2012, U.S. Provisional Patent Application No. 61 / 615,618, filed March 26, 2012, U.S. Provisional Patent Application No. 61 / 680,098, filed August 6, 2012, U.S. Patent Application No. 14 / 358,972, filed May 16, 2014, and International Application No. PCT / US2012 / 065691, filed November 16, 2012, the entire contents of each of which are hereby incorporated by reference herein.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety. The ASCII copy, created on July 8, 2016, is named 121301-03020_SL.txt and is 68,289 bytes in size. [Background technology]

[0004] 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 the 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 beta peptide, and neuropeptide Y. (See Non-Patent Document 1).

[0005] TTR is a tetramer of four identical 127 amino acid subunits (monomers) enriched in a beta-sheet structure. Each monomer has two four-stranded beta-sheets and a prolate spheroidal shape. Antiparallel beta-sheet interactions link the monomers into dimers. A short loop from each monomer forms the major dimer-dimer interaction. These two loop pairs separate the opposing convex beta-sheets of the dimer, forming an internal channel.

[0006] The liver is the major site of TTR expression. Other prominent sites of expression include the choroid plexus, retina (especially the retinal pigment epithelium), and pancreas.

[0007] Transthyretin is one of at least 27 different types of proteins that are precursor proteins in the formation of amyloid fibrils. See (Non-Patent Document 2). Extracellular deposition of amyloid fibrils in organs and tissues is a hallmark of amyloidosis. Amyloid fibrils consist of misfolded protein aggregates that can result from either excessive production of precursor proteins or specific mutations in the precursor proteins. 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, (Non-Patent Document 3).

[0008] 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., their names begin with "A" for amyloid, followed by the abbreviation for the precursor protein, e.g., ATTR for amyloidogenic transthyretin (ibid.).

[0009] There are numerous TTR-related diseases, most of which are amyloid diseases. Normal-sequence TTR is associated with cardiac amyloidosis in elderly people, 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 referred to as familial amyloid polyneuropathy (FAP). When the heart is primarily involved but not the nervous system, the disease is referred to as 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 (a non-amyloid disease thought to be secondary to increased association of TTR with thyroxine due to mutant TTR molecules with increased affinity for thyroxine). See, e.g., (Non-Patent Document 4).

[0010] Abnormal amyloidogenic proteins can be either inherited or acquired through somatic mutations (Non-Patent Document 2). Transthyretin-associated ATTR is the most common form of hereditary systemic amyloidosis (Non-Patent Document 5). 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 cause systemic amyloid deposition, particularly involving the peripheral nervous system, but some mutations are associated with cardiomyopathy or vitreous opacities (ibid.).

[0011] The V30M mutation is the most common TTR mutation. See, for example, (Non-Patent Document 5). The V122I mutation is carried by 3.9% of the African American population and is the most common cause of FAC (Non-Patent Document 6). It is estimated that SSA affects more than 25% of the population over 80 years of age (Non-Patent Document 7). [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Liz, MAet al. (2010) IUBMB Life,62(6):429-435 [Non-patent document 2] Guan,J.et al.(Nov.4,2011)Current perspectives on cardiac amyloidosis,Am J Physiol Heart Circ Physiol,doi:10.1152 / ajpheart.00815.2011 [Non-patent document 3] Saraiva MJM(2002)Expert Reviews in Molecular Medicine,4(12):1-11 [Non-patent document 4] Moses et al.(1982)J.Clin.Invest.,86,2025-2033 [Non-Patent Document 5] Lobato, L. (2003) J. Nephrol., 16:438-442 [Non-patent document 6] Jacobson, DRet al. (1997) N. Engl. J. Med. 336(7):466-73 [Non-Patent Document 7] Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. USA87(7):2843-5 Summary of the Invention [Problem to be solved by the invention]

[0013] Thus, there is a need in the art for effective treatments for TTR-related diseases. [Means for solving the problem]

[0014] The present invention provides RNAi agents, such as double-stranded RNAi agents, and compositions that target transthyretin (TTR) gene.The present invention also provides a method for inhibiting the expression of TTR and a method for treating or preventing TTR-related diseases in subjects using the RNAi agents, such as double-stranded RNAi agents, of the present invention.At least in part, the present invention is based on the discovery herein that RNAi agents, in which substantially all of the nucleotides on the sense strand and substantially all of the nucleotides on the antisense strand are modified nucleotides, and which comprise 8 or less 2'-fluoro modifications on the sense strand, 6 or less 2'-fluoro modifications on the antisense strand, two phosphorothioate bonds at the 5' end of the sense strand, two phosphorothioate bonds at the 5' end of the antisense strand, and ligands, such as GalNAc3 ligands, are effective in stopping the activity of TTR gene.These agents unexpectedly show enhanced TTR gene silencing activity. Without intending to be bound by theory, it is believed that the combination or subcombination of the aforementioned modifications and specific target sites in these RNAi agents confers improved efficacy, stability, potency, and durability to the RNAi agents of the present invention.

[0015] Accordingly, in one aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of transthyretin (TTR) in a cell, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, wherein the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0016] In one embodiment, the double-stranded RNAi agent has formula (IIIe): Sense: 5'-Na -YYY-N b -3' Antisense: 3'-n p '-N a '-Y'Y'Y'-N b '-5' (IIIe) (In the formula, n p ' is a two nucleotide overhang, and n p 'Each internal nucleotide is linked to the adjacent nucleotide via a phosphorothioate bond; each N a , N b , N a ' and N b ' independently represent an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified or a combination thereof, each sequence containing at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0017] In one embodiment, the YYY motif is present at or near the cleavage site on the sense strand, hi one embodiment, the Y'Y'Y' motif is present at positions 11, 12, and 13 from the 5' end on the antisense strand.

[0018] In one embodiment, the Y nucleotide comprises a 2'-fluoro modification.

[0019] In one embodiment, the Y' nucleotide comprises a 2'-O-methyl modification.

[0020] The double-stranded region can be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0021] Each strand of the double-stranded RNAi agent can have 15-30 nucleotides or 19-30 nucleotides.

[0022] In one embodiment, the modification on the nucleotide is selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic, and combinations thereof.

[0023] In one embodiment, the modification on the nucleotide is a 2'-O-methyl or a 2'-fluoro modification.

[0024] The sense strand may include no more than 7 2'-fluoro modifications, no more than 6 2'-fluoro modifications, no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 3 2'-fluoro modifications, or no more than 2 2'-fluoro modifications.

[0025] The antisense strand may include no more than 5 2'-fluoro modifications, no more than 4 2'-fluoro modifications, no more than 3 2'-fluoro modifications, or no more than 2 2'-fluoro modifications.

[0026] In one embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate or a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic at the 5' nucleotide of the antisense strand.

[0027] In one embodiment, the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

[0028] In one embodiment, the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. [ka] is.

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

[0030] In one embodiment, the double-stranded RNAi agent is conjugated to a ligand as shown in the following schematic diagram. [ka] (wherein X is O or S)

[0031] In one embodiment, the antisense strand is 5'-usCfsuugguuacaugAfaaucccasusc-3' (SEQ ID NO: 6), 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), 5'-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 8), and 5'-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 9) wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage; and VP is a 5'-phosphate mimetic. The nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:

[0032] In one embodiment, the sense and antisense strands are 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugguuacaugAfaaucccasusc-3' (SEQ ID NO: 6); 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7); 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 8); and 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 9) wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage; and VP is a 5'-phosphate mimetic. In another embodiment, the sense and antisense strands comprise the nucleotide sequences 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage. In yet another embodiment, the RNAi agent is selected from the group of any one of the RNAi agents listed in any one of Tables 1 and 3. In yet another embodiment, the RNAi agent is AD-65492.

[0033] In one aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of transthyretin (TTR) in a cell, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region fully complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, wherein the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0034] In one embodiment, the double-stranded RNAi agent has formula (IIIe): Sense: 5'-N a -YYY-N b-3' Antisense: 3'-n p '-N a '-Y'Y'Y'-N b '-5' (IIIe) (In the formula, n p ' is a two nucleotide overhang, and n p 'Each internal nucleotide is linked to an adjacent nucleotide via a phosphorothioate bond; each N a , N b , N a ' and N b ' independently represent an oligonucleotide sequence comprising 8-10 nucleotides that are either modified or unmodified or a combination thereof, each sequence comprising at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications. is expressed by

[0035] The present invention also provides cells comprising the double-stranded RNAi agents of the present invention, cells comprising the vectors of the present invention, and pharmaceutical compositions comprising the double-stranded RNAi agents of the present invention or the vectors of the present invention.

[0036] In one embodiment, the double-stranded RNAi agent is administered in an unbuffered solution, such as saline or water.

[0037] In another embodiment, double-stranded RNAi agent is administered with a buffer solution.In one embodiment, the buffer solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.In another embodiment, the buffer solution is phosphate buffered saline (PBS).

[0038] In another aspect, the present invention provides a method for inhibiting expression of transthyretin (TTR) in a cell, the method comprising: (a) contacting a cell with a double-stranded RNAi agent of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention; and (b) maintaining the cell produced in step (a) for a time sufficient to allow degradation of mRNA transcripts of the TTR gene, thereby inhibiting expression of the TTR gene in the cell.

[0039] In one embodiment, the cell is within a subject.

[0040] In one embodiment, the subject is a human.

[0041] In one embodiment, the subject has a TTR-related disorder.

[0042] In one embodiment, expression of TTR is inhibited by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0043] In yet another aspect, the present invention provides a method for treating a subject having a transthyretin (TTR)-related disorder by administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby treating the subject.

[0044] In yet another aspect, the present invention provides a method for prophylactically treating a subject at risk of developing a transthyretin (TTR)-associated disorder by administering to the subject a prophylactically effective amount of a double-stranded RNAi agent of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby prophylactically treating the subject.

[0045] In a further aspect, the present invention provides a method of treating a subject having a transthyretin (TTR)-related disorder, the method comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand being about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand being modified nucleotides, the sense strand comprising no more than eight 2'-fluoro modifications; the antisense strand comprising no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprising two phosphorothioate linkages at their 5' termini; and the sense strand being conjugated to at least one ligand.

[0046] In a further aspect, the present invention provides a method for prophylactically treating a subject at risk of developing a transthyretin (TTR)-associated disorder, comprising administering to the subject a prophylactically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand being about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand being modified nucleotides, the sense strand comprising no more than eight 2'-fluoro modifications; the antisense strand comprising no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprising two phosphorothioate linkages at their 5' termini; and the sense strand being conjugated to at least one ligand.

[0047] In one aspect, the present invention provides a method for reducing, slowing, or halting the Neuropathy Impairment Score (NIS) or revised NIS (mNIS+7) in a subject with a transthyretin (TTR)-related disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby reducing, slowing, or halting the Neuropathy Impairment Score (NIS) or revised NIS (mNIS+7) in the subject.

[0048] In a further aspect, the present invention provides a method for reducing, slowing, or halting the Neuropathy Impairment Score (NIS) or revised NIS (mNIS+7) in a subject with a transthyretin (TTR)-related disorder. The method includes administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to SEQ ID NO: 2, each strand being about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0049] In one aspect, the present invention provides a method for increasing 6-minute walk test (6MWT) in a subject with a transthyretin (TTR)-related disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention, thereby increasing the 6-minute walk test (6MWT) in the subject.

[0050] In a further aspect, the present invention provides a method for increasing 6-minute walk test (6MWT) performance in a subject with a transthyretin (TTR)-associated disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to SEQ ID NO: 2, each strand being about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand being modified nucleotides, the sense strand comprising no more than eight 2'-fluoro modifications; the antisense strand comprising no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprising two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0051] In one embodiment, the double-stranded RNAi agent has formula (IIIe): Sense: 5'-N a -YYY-N b -3' Antisense: 3'-n p '-N a '-Y'Y'Y'-N b '-5' (IIIe) (In the formula, n p ' is a two nucleotide overhang, and n p 'Each internal nucleotide is linked to an adjacent nucleotide via a phosphorothioate bond; each N a , N b , N b and N b ' independently represent an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified or a combination thereof, each sequence containing at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0052] In one embodiment, the subject is a human.

[0053] In one embodiment, the subject is a subject suffering from a TTR-related disease. In another embodiment, the subject is a subject at risk of developing a TTR-related disease. In one embodiment, a subject at risk of developing a TTR-related disease, or a subject with a family history of a TTR-related disease, or a subject with signs or symptoms suggestive of developing TTR amyloidosis, has a TTR gene mutation associated with the development of a TTR-related disease.

[0054] In one embodiment, the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia.

[0055] In one embodiment, the subject has TTR-related amyloidosis, and the method reduces amyloid TTR deposits in the subject.

[0056] In one embodiment, the double-stranded RNAi agent is administered to the subject by the administration means selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof.In another embodiment, the double-stranded RNAi agent is administered to the subject by subcutaneous, intramuscular or intravenous administration.In yet another embodiment, the double-stranded RNAi agent is administered to the subject by subcutaneous administration.

[0057] In one embodiment, the method further comprises assessing the level of mRNA expression or TTR protein expression in a sample from the subject.

[0058] In one embodiment, administration of the double-stranded RNAi agent does not result in an inflammatory response in the subject as assessed based on the level of a cytokine or chemokine selected from the group consisting of G-CSF, IFN-γ, IL-10, IL-12(p70), IL1β, IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, and any combination thereof in a sample from the subject.

[0059] In one aspect, the invention provides a method of treating a subject having a transthyretin (TTR)-related disorder. The method includes administering to a subject a dose of about 12.5 mg to about 200 mg (e.g., about 12.5 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg) of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, wherein the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0060] In another aspect, the present invention provides a method for prophylactically treating a subject at risk of developing a transthyretin (TTR)-associated disorder. The method includes administering to a subject a dose of about 12.5 mg to about 200 mg (e.g., about 12.5 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg) of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, wherein the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0061] In one aspect, the present invention provides a method for treating a subject having a transthyretin (TTR)-related disorder, comprising administering to the subject a double-stranded RNAi agent at a dose of about 0.15 mg / kg to about 2.5 mg / kg (e.g., about 0.15 mg / kg, about 0.3 mg / kg, about 0.6 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 2 mg / kg, about 2.5 mg / kg, or about 3 mg / kg), wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, and the antisense strand is SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA- 3') of the sense strand, each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5'-terminus; and the sense strand is conjugated to at least one ligand.

[0062] In one aspect, the present invention provides a method for prophylactically treating a subject at risk of developing a transthyretin (TTR)-associated disorder. The method includes administering to the subject a double-stranded RNAi agent at a dose of about 0.15 mg / kg to about 2.5 mg / kg (e.g., about 0.15 mg / kg, about 0.3 mg / kg, about 0.6 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 2 mg / kg, about 2.5 mg / kg, or about 3 mg / kg), wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, and the antisense strand is SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA- 3') of the sense strand, each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5'-terminus; and the sense strand is conjugated to at least one ligand.

[0063] In another aspect, the present invention provides a method for reducing, slowing, or halting the Neuropathy Impairment Score (NIS) or revised NIS (mNIS+7) in a subject with a transthyretin (TTR)-related disorder. The method includes administering to the subject a double-stranded RNAi agent at a dose of about 0.15 mg / kg to about 2.5 mg / kg (e.g., about 0.15 mg / kg, about 0.3 mg / kg, about 0.6 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 2 mg / kg, about 2.5 mg / kg, or about 3 mg / kg), wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, and the antisense strand is SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA- 3') of the sense strand, each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5'-terminus; and the sense strand is conjugated to at least one ligand.

[0064] In yet another aspect, the present invention provides a method for increasing 6-minute walk test (6MWT) in a subject with a transthyretin (TTR)-related disorder. The method includes administering to the subject a double-stranded RNAi agent at a dose of about 0.15 mg / kg to about 2.5 mg / kg (e.g., about 0.15 mg / kg, about 0.3 mg / kg, about 0.6 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 2 mg / kg, about 2.5 mg / kg, or about 3 mg / kg), wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, and the antisense strand is SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA- 3') of the sense strand, each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5'-terminus; and the sense strand is conjugated to at least one ligand.

[0065] In one aspect, the present invention provides a method for treating a subject with a transthyretin (TTR)-related disorder. The method includes administering a double-stranded RNAi agent in an amount of about 10 mg to about 600 mg, about 25 mg to about 500 mg, about 50 mg to about 500 mg, or about 80 mg to about 500 mg, about 25 mg to about 300 mg, about 50 mg to about 300 mg, or about 80 mg to about 300 mg (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, about 125, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 610, about 620, about 630, about 640, about 650, about 660 60, about 170, about 175, about 180, about 190, about 200, about 210, about 220, about 225, about 230, about 240, about 250 mg, about 260, about 270, about 275, about 280, about 290, about 300, about 310, about 320, about 325, about 330, about 340, about 350, about 360, about 370, about 375, about 380, about 390, about 400, about 410, about 420, about 425, about 430, about 440, about 450 mg, about 460, about 470, the antisense strand comprises a region complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand comprises eight or fewer 2'-fluoro modifications; the antisense strand comprises six or fewer 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0066] In one aspect, the present invention provides a method for prophylactically treating a subject at risk of developing a transthyretin (TTR)-related disorder. The method includes administering a double-stranded RNAi agent at a dose of about 10 mg to about 600 mg, about 25 mg to about 500 mg, about 50 mg to about 500 mg, or about 80 mg to about 500 mg, about 25 mg to about 300 mg, about 50 mg to about 300 mg, or about 80 mg to about 300 mg (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, about 125, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 610, about 620, about 630, about 640, about 65 60, about 170, about 175, about 180, about 190, about 200, about 210, about 220, about 225, about 230, about 240, about 250 mg, about 260, about 270, about 275, about 280, about 290, about 300, about 310, about 320, about 325, about 330, about 340, about 350, about 360, about 370, about 375, about 380, about 390, about 400, about 410, about 420, about 425, about 430, about 440, about 450 mg, about 460, about 470, the antisense strand comprises a region complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, the sense strand comprises eight or fewer 2'-fluoro modifications; the antisense strand comprises six or fewer 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0067] In another aspect, the present invention provides a method for reducing, slowing, or halting the Neuropathy Impairment Score (NIS) or revised NIS (mNIS+7) in a subject with a transthyretin (TTR)-related disorder. The method includes administering a double-stranded RNAi agent in an amount of about 10 mg to about 600 mg, about 25 mg to about 500 mg, about 50 mg to about 500 mg, or about 80 mg to about 500 mg, about 25 mg to about 300 mg, about 50 mg to about 300 mg, or about 80 mg to about 300 mg (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, about 125, about 130, about 140, about 150, about 160, about 170, about 175, about 180, about 190, about 200, about 210, about 220, about 225, about 230, about 240, about 250, about 260, about 270, about 275, about 280, about 290, about 300, about 310, about 320, about 325, about 330, about 340, about 350, about 360, about 370, about 375, about 380, about 390, about 400, about 410, about 420, about 425, about 430, about 440, about 450, about 460, about 470, about 480 the antisense strand comprises about 14 to about 30 nucleotides in length, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0068] In yet another aspect, the present invention provides a method for increasing 6-minute walk test (6MWT) in a subject with a transthyretin (TTR)-related disorder. The method includes administering a double-stranded RNAi agent at a dose of about 10 mg to about 600 mg, about 25 mg to about 500 mg, about 50 mg to about 500 mg, or about 80 mg to about 500 mg, about 25 mg to about 300 mg, about 50 mg to about 300 mg, or about 80 mg to about 300 mg (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90, about 100, about 110, about 120, about 125, about 130, about 140, about 150, about 160, about 170, about 175, about 180, about 190, about 200, about 210, about 220, about 225, about 230, about 240, about 250, about 260, about 270, about 275, about 280, about 290, about 300, about 310, about 320, about 325, about 330, about 340, about 350, about 360, about 370, about 375, about 380, about 390, about 400, about 410, about 420, about 425, about 430, about 440, about 450, about 460, about 470, about 480 the antisense strand comprises about 14 to about 30 nucleotides in length, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand comprises no more than eight 2'-fluoro modifications; the antisense strand comprises no more than six 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at their 5' termini; and the sense strand is conjugated to at least one ligand.

[0069] In one embodiment, the double-stranded RNAi agent has formula (IIIe): Sense: 5'-N a -YYY-N b -3' Antisense: 3'-n p '-Na '-Y'Y'Y'-N b '-5' (IIIe) (In the formula, n p ' is a two nucleotide overhang, and n p 'Each internal nucleotide is linked to an adjacent nucleotide via a phosphorothioate bond; each N a , N b , N b and N b ' independently represent an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified or a combination thereof, each sequence containing at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0070] In one embodiment, the antisense strand is 5'-usCfsuugguuacaugAfaaucccasusc-3' (SEQ ID NO: 6), 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), 5'-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 8), and 5'-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 9) wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; s is a phosphorothioate linkage; and VP is a 5'-phosphate mimetic. The nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:

[0071] In one embodiment, the sense and antisense strands are 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugguuacaugAfaaucccasusc-3' (SEQ ID NO: 6); 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7); 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 8); and 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 9) wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage; and VP is a 5'-phosphate mimetic.

[0072] In one embodiment, the sense and antisense strands are 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7) wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

[0073] A dose of the double-stranded RNAi agent may be administered to the subject about once every 4 weeks, 5 weeks, 6 weeks, 8 weeks, or quarterly.

[0074] The dose of double-stranded RNAi agent may be administered to the subject about once every 4 weeks, 5 weeks, 6 weeks, 8 weeks, or quarterly.

[0075] In one embodiment, the double-stranded RNAi agent is administered to the subject about once every quarter.

[0076] In one embodiment, the double-stranded RNAi agent is administered chronically to the subject.

[0077] In one embodiment, the subject is a human.

[0078] In one embodiment, the subject is a subject suffering from a TTR-related disease. In another embodiment, the subject is a subject at risk of developing a TTR-related disease. In one embodiment, a subject at risk of developing a TTR-related disease, or a subject with a family history of a TTR-related disease, or a subject with signs or symptoms suggestive of developing TTR amyloidosis, has a TTR gene mutation associated with the development of a TTR-related disease.

[0079] In one embodiment, the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia.

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

[0081] In one embodiment, double-stranded RNAi agent is administered to subject by administration means selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof.In another embodiment, double-stranded RNAi agent is administered to subject via subcutaneous, intramuscular or intravenous administration.In yet another embodiment, double-stranded RNAi agent is administered to subject via subcutaneous administration, for example, via self-administration, for example, via pre-filled syringe or auto-injector syringe.

[0082] In one embodiment, the method further comprises assessing the level of mRNA expression or TTR protein expression in a sample from the subject.

[0083] In one embodiment, administration of the double-stranded RNAi agent does not result in an inflammatory response in the subject as assessed based on the level of a cytokine or chemokine selected from the group consisting of G-CSF, IFN-γ, IL-10, IL-12(p70), IL1β, IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, and any combination thereof in a sample from the subject.

[0084] In one embodiment, an agent suitable for use in the methods of the invention is AD-65492. AD-65492 may be administered chronically to a subject every 4 weeks, every 5 weeks, or every 6 weeks, or quarterly.

[0085] In one aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent for use in inhibiting expression of transthyretin (TTR) in a cell, the agent comprising a sense strand complementary to an antisense strand, wherein the sense and antisense strands comprise a nucleotide sequence selected from the group consisting of any of the nucleotide sequences in Table 5.

[0086] In another aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent for use in inhibiting expression of transthyretin (TTR) in a cell, the agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region of complementarity comprising at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any one of the antisense sequences in Table 6, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides; and the sense strand is conjugated to at least one ligand.

[0087] The sense and antisense strands may comprise a nucleotide sequence selected from the group consisting of any of the nucleotide sequences in Table 6 or Table 7.

[0088] In one aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent for use in inhibiting expression of transthyretin (TTR) in a cell, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

[0089] In another aspect, the present invention provides a method for treating a subject suffering from a TTR-related disease, comprising administering to the subject a double-stranded RNAi agent at a dose of about 50 mg to about 300 mg, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, the sense strand comprising the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage, thereby treating the subject suffering from a TTR-related disease.

[0090] In yet another aspect, the present invention provides a method for prophylactically treating a subject at risk of developing a TTR-related disorder, comprising administering to the subject a double-stranded RNAi agent at a dose of about 50 mg to about 300 mg, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, the sense strand comprising the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage, thereby prophylactically treating the subject at risk of developing a TTR-related disorder.

[0091] In one aspect, the present invention provides a method for reducing, slowing, or halting the Neuropathy Impairment Score (NIS) or modified NIS (mNIS+7) in a subject suffering from or at risk of developing a TTR-related disorder. The method includes administering to a subject a dose of about 50 mg to about 300 mg of a double-stranded RNAi agent, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage, thereby reducing, slowing, or halting the Neuropathy Impairment Score (NIS) or revised NIS (mNIS+7) in the subject.

[0092] In another aspect, the present invention provides a method of increasing the 6-minute walk test (6MWT) in a subject suffering from or at risk of developing a TTR-related disorder. The method includes administering to a subject a dose of about 50 mg to about 300 mg of a double-stranded RNAi agent, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), where a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage, thereby increasing 6-minute walk test (6MWT) in a subject suffering from or at risk of developing a TTR-related disorder.

[0093] The present invention is further illustrated by the following detailed description and drawings. [Brief explanation of the drawings]

[0094] [Figure 1] 1 is a graph depicting the stability of the indicated RNAi agents in a 24-hour tritosome stability assay. [Figure 2] FIG. 2A is a graph showing the stability of the indicated RNAi agents in a 24-hour rat cytosolic stability assay, and FIG. 2B is a graph showing the stability of the indicated RNAi agents in a 24-hour tritosomal stability assay. [Figure 3] 1 is a graph showing TTR protein suppression in transgenic mice expressing the V30M mutant of human TTR (V30M hTTR) following administration of a single subcutaneous dose of 1 mg / kg of the indicated RNAi agent. [Figure 4] 1 is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M following administration of a single subcutaneous dose of 2.5 mg / kg of the indicated RNAi agent. [Figure 5] 1 is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M after administration of AD-65492 at a weekly dose of 2 mg / kg for 3 weeks (QW×3). [Figure 6-1] Figure 6A is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M after subcutaneous administration of the indicated RNAi agent at a monthly dose of 0.3 mg / kg for four months (QM x 4 @ 0.3 mg / kg), Figure 6B is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M after subcutaneous administration of the indicated RNAi agent at a monthly dose of 1 mg / kg for four months (QM x 4 @ 1 mg / kg), and Figure 6C is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M after subcutaneous administration of the indicated RNAi agent at a monthly dose of 3 mg / kg for four months (QM x 4 @ 3 mg / kg). [Figure 6-2]Figure 6A is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M after subcutaneous administration of the indicated RNAi agent at a monthly dose of 0.3 mg / kg for four months (QM x 4 @ 0.3 mg / kg), Figure 6B is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M after subcutaneous administration of the indicated RNAi agent at a monthly dose of 1 mg / kg for four months (QM x 4 @ 1 mg / kg), and Figure 6C is a graph showing TTR protein suppression in transgenic mice expressing hTTR V30M after subcutaneous administration of the indicated RNAi agent at a monthly dose of 3 mg / kg for four months (QM x 4 @ 3 mg / kg). [Figure 7] 1 shows the study design for subcutaneous administration of AD-65492 and AD-66017 to cynomolgus monkeys. [Figure 8-1] Figure 8A is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 0.3 mg / kg of the indicated RNAi agent. Figure 8B is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 1 mg / kg of AD-65492, a single subcutaneous dose of 1 mg / kg of AD-66017, or a single subcutaneous dose of 2.5 mg / kg of AD-51547. Figure 8C is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 3 mg / kg of AD-65492, a single subcutaneous dose of 3 mg / kg of AD-66017, or a single subcutaneous dose of 5 mg / kg of AD-51547. [Figure 8-2] Figure 8A is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 0.3 mg / kg of the indicated RNAi agent. Figure 8B is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 1 mg / kg of AD-65492, a single subcutaneous dose of 1 mg / kg of AD-66017, or a single subcutaneous dose of 2.5 mg / kg of AD-51547. Figure 8C is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 3 mg / kg of AD-65492, a single subcutaneous dose of 3 mg / kg of AD-66017, or a single subcutaneous dose of 5 mg / kg of AD-51547. [Figure 8-3] Figure 8A is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 0.3 mg / kg of the indicated RNAi agent. Figure 8B is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 1 mg / kg of AD-65492, a single subcutaneous dose of 1 mg / kg of AD-66017, or a single subcutaneous dose of 2.5 mg / kg of AD-51547. Figure 8C is a graph showing TTR protein suppression in cynomolgus monkeys after administration of a single subcutaneous dose of 3 mg / kg of AD-65492, a single subcutaneous dose of 3 mg / kg of AD-66017, or a single subcutaneous dose of 5 mg / kg of AD-51547. [Figure 9-1] Figure 9A is a graph showing TTR protein suppression in cynomolgus monkeys after administration of AD-65492 at a monthly subcutaneous dose of 1 mg / kg for 4 months (QM x 4), AD-66017 at a monthly subcutaneous dose of 1 mg / kg for 4 months (QM x 4), or AD-51547 at a daily dose of 5 mg / kg for 5 days followed by a weekly dose of 5 mg / kg for 4 weeks (QD x 5, QW x 4). Figure 9B is a graph showing TTR protein suppression in cynomolgus monkeys after administration of the indicated RNAi agents at a monthly subcutaneous dose of 3 mg / kg for 4 months (QM x 4). [Figure 9-2] Figure 9A is a graph showing TTR protein suppression in cynomolgus monkeys after administration of AD-65492 at a monthly subcutaneous dose of 1 mg / kg for 4 months (QM x 4), AD-66017 at a monthly subcutaneous dose of 1 mg / kg for 4 months (QM x 4), or AD-51547 at a daily dose of 5 mg / kg for 5 days followed by a weekly dose of 5 mg / kg for 4 weeks (QD x 5, QW x 4). Figure 9B is a graph showing TTR protein suppression in cynomolgus monkeys after administration of the indicated RNAi agents at a monthly subcutaneous dose of 3 mg / kg for 4 months (QM x 4). [Figure 10-1] 1 is a graph showing maintenance of TTR suppression in cynomolgus monkeys with monthly subcutaneous administration of 1 mg / kg doses of AD-65492 for 4 months (QM×4; solid line) compared with TTR suppression after a single subcutaneous administration of AD-65492 at 1 mg / kg (dashed line). [Figure 10-2]1 is a graph showing the additive effect of monthly subcutaneous administration of AD-66017 at 1 mg / kg doses for 4 months (QM×4; solid line) on TTR protein suppression compared with a single subcutaneous dose of AD-66017 at 1 mg / kg (dashed line) in cynomolgus monkeys. [Figure 11] 1 is a graph showing sustained serum TTR suppression in cynomolgus monkeys after monthly subcutaneous administration of AD-65492 at a dose of 1 mg / kg for 4 months (QM×4) or monthly subcutaneous administration of AD-65492 at a dose of 3 mg / kg for 4 months (QM×4) compared to a single subcutaneous administration of AD-65492 at a dose of 1 mg / kg or a single subcutaneous administration of AD-65492 at a dose of 0.3 mg / kg. [Figure 12] 1 shows the study design for subcutaneous administration of AD-65492 to cynomolgus monkeys. [Figure 13] 1 is a graph showing robust serum TTR suppression in cynomolgus monkeys after monthly subcutaneous administration of AD-65492 at a dose of 0.3 mg / kg for 6 months (QM x 6), or monthly subcutaneous administration of AD-65492 at a dose of 0.6 mg / kg for 6 months (QM x 6), or a single initial 1 mg / kg dose of AD-65492 (QM x 1) followed by monthly 0.3 mg / kg doses of AD-65492 for 5 months (QM x 5) starting on day 28 after the initial dose. DETAILED DESCRIPTION OF THE INVENTION

[0095] The present invention provides RNAi agents, e.g., double-stranded RNAi agents, and compositions that target the transthyretin (TTR) gene. The present invention also provides methods for inhibiting TTR expression and treating or preventing TTR-related diseases in subjects using the RNAi agents, e.g., double-stranded RNAi agents, of the present invention. The present invention is based, at least in part, on the discovery that the RNAi agent comprising substantially all of the nucleotides on the sense strand and substantially all of the nucleotides on the antisense strand are modified nucleotides, and 8 or less 2'-fluoro modifications on the sense strand (for example, 7 or less 2'-fluoro modifications, 6 or less 2'-fluoro modifications, 5 or less 2'-fluoro modifications, 4 or less 2'-fluoro modifications, 5 or less 2'-fluoro modifications, 4 or less 2'-fluoro modifications, 3 or less 2'-fluoro modifications, or 2 or less 2'-fluoro modifications) and 6 or less 2'-fluoro modifications on the antisense strand (for example, 5 or less 2'-fluoro modifications, 4 or less 2'-fluoro modifications, 3 or less 2'-fluoro modifications, or 2 or less 2'-fluoro modifications), two phosphorothioate bonds at the 5' end of the sense strand, two phosphorothioate bonds at the 5' end of the antisense strand, and a ligand, such as GalNAc3 ligand, is shown herein to be effective for selectively silencing the activity of TTR gene.These agents unexpectedly show enhanced TTR gene silencing activity. Without intending to be bound by theory, it is believed that the combination or subcombination of the aforementioned modifications and specific target sites in these RNAi agents confers improved efficacy, stability, potency, and durability to the RNAi agents of the present invention.

[0096] The following detailed description discloses methods for making and utilizing compositions containing iRNAs that selectively inhibit TTR gene expression, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from inhibition and / or reduction of TTR gene expression.

[0097] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values ​​for a parameter is listed, values ​​and ranges intermediate to the listed values ​​are also intended to be part of the invention.

[0098] 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. By way of example, "an element" means one element or two or more elements, such as, for example, a plurality of elements.

[0099] The term "including" is used herein to mean, and is used interchangeably with, the term "including but not limited to."

[0100] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0101] The term "about" is used herein to mean within a typical range accepted in the art. For example, "about" can be understood as within 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 a range, it is understood that "about" can modify each of the series of numbers or ranges.

[0102] 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 it 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 and mutant TTR proteins can form amyloid fibrils that aggregate into extracellular deposits, causing 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 by Dickson, P. W. et al. (1985) J. Biol. Chem. 260(13) 8214-8219. The X-ray crystal structure of human TTR is described by Blake, C. C. et al. (1974) J. Mol. Biol. 88, 1-12. The sequence of human TTR mRNA transcripts can be found in the National Center for Biotechnology Information (NCBI) RefSeq accession number NM_000371 (e.g., SEQ ID NOs: 1 and 5). The sequence of mouse TTR mRNA can be found in RefSeq accession number NM_013697.2, and the sequence of rat TTR mRNA can be found in RefSeq accession number NM_012681.1. Further examples of TTR mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0103] 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, including mRNA, which is an RNA processing product of the primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage at or near the portion of the nucleotide sequence of the mRNA molecule formed during transcription of the TTR gene. In one embodiment, the target sequence is located within the protein-coding region of the TTR gene. In another embodiment, the target sequence is located within the 3'UTR of the TTR gene.

[0104] The target sequence may be about 9-36 nucleotides in length, such as about 15-30 nucleotides in length. For example, the target sequence may be 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-2 The target sequence may be about 15 to 30 nucleotides in length, such as 6, 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 some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also considered to be part of this invention.

[0105] In some embodiments of the invention, the target sequence of the TTR gene comprises nucleotides 615 to 637 of SEQ ID NO: 1 or nucleotides 505 to 527 of SEQ ID NO: 5 (i.e., 5'-GATGGGATTTCATGTAACCAAGA-3'; SEQ ID NO: 4).

[0106] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide that comprises a chain of nucleotides described by a referenced sequence, using standard nucleotide nomenclature.

[0107] "G", "C", "A", "T" and "U" generally represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as base, respectively.However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or alternative replacement moieties, as will be further detailed below (see, for example, Table 2).Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moieties.As a non-limiting example, the nucleotide that contains inosine as base can base pair with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be replaced with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA of the present invention. In another example, adenine and cytosine can be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured herein.

[0108] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" are used interchangeably herein and refer to an agent that contains RNA, as defined herein, and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, TTR gene expression in a cell, e.g., a cell in a subject, e.g., a mammalian subject.

[0109] In one embodiment, the RNAi agent of the present invention includes a single-stranded RNA that interacts with a target RNA sequence, such as a TTR target mRNA sequence, to induce 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 by a type III endonuclease known as Dicer, resulting in short double-stranded interfering RNAs (siRNAs) containing sense and antisense strands (Sharp et al. (2001) Genes Dev. 15:485). The Dicer RNase III-like enzyme processes these dsRNAs to produce short interfering RNAs of 19-23 base pairs, characterized by a 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When binding to appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev.15:188). Thus, in one aspect, the present invention relates to single-stranded siRNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced inside cells and promotes the formation of RISC complex to silence target gene, i.e., TTR gene. Therefore, the term "siRNA" is also used herein to refer to RNAi as described above.

[0110] In another embodiment, the RNAi agent may be a single-stranded RNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2 and then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and chemically modified. The design and testing of single-stranded RNAs 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 antisense nucleotide sequence described herein may be used as a single-stranded siRNA as described herein, or chemically modified as described in Lima et al., (2012) Cell 150:883-894.

[0111] In another embodiment, 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 RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a ribonucleic acid molecule complex having a double-stranded structure, comprising two antiparallel, substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations with respect to the target RNA, i.e., the TTR gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) causes target RNA degradation, such as mRNA, through a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0112] Generally, the majority of the nucleotides in each strand of dsRNA molecule are ribonucleotides, but as described herein, each strand or both strands can also comprise one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.Furthermore, as used herein, " RNAi agent " can comprise ribonucleotides with chemical modification; RNAi agent can comprise substantial modification in multiple nucleotides.

[0113] As used herein, the term "modified nucleotide" refers to a nucleotide that has, independently, a modified sugar moiety, a modified internucleotide bond, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses, for example, the substitution, addition, or removal of a functional group or atom to an internucleoside bond, a sugar moiety, or a nucleobase. Modifications suitable for use in the agent of the present invention include all types of modifications disclosed herein or known in the art. Any such modifications are encompassed by "RNAi agent" when used in siRNA-type molecules for the purposes of this application and claims.

[0114] The double-stranded region may be of any length that allows for specific degradation of the desired target RNA through the RISC pathway, and may range from about 9 to 36 base pairs in length, for example, about 15 to 30 base pairs in length, such as 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, for example, about 15 to 30, 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, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, and 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

[0115] The two strands forming the double-stranded structure may be different portions of a larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of a single larger molecule and are thus connected by an uninterrupted stretch of nucleotides between the 3' end of one strand and the 5' end of the other strand, the combined RNA strands are referred to as "hairpin loops." A hairpin loop may contain at least one unpaired nucleotide; in some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, or at least 23 or more unpaired nucleotides. In some embodiments, a hairpin loop may be 10 or fewer nucleotides long. In some embodiments, a hairpin loop may be 8 or fewer unpaired nucleotides long. In some embodiments, a hairpin loop may be 4 to 10 unpaired nucleotides long. In some embodiments, a hairpin loop may be 4 to 8 nucleotides long.

[0116] When the two substantially complementary strands of dsRNA are constituted by another RNA molecule, these molecules can be covalently linked, but do not necessarily have to be.When the two strands are covalently linked by means other than an uninterrupted nucleotide chain between the 3'-end of one strand that forms a double-stranded structure and the 5'-end of each other strand, the linked structure is called "linker".RNA strands can have the same or different nucleotide numbers.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs that exist in the double strand.In addition to the double-stranded structure, RNAi can also include one or more nucleotide overhangs.

[0117] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which is 24-30 nucleotides in length, that interacts with a target RNA sequence, e.g., a TTR target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, 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). The Dicer RNase III-like enzyme processes the dsRNA to produce short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment of the RNAi agent, at least one strand comprises a 3' overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15. In still other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0118] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which contains 19-23 nucleotides, that interacts with the TTR RNA sequence to induce cleavage of the target RNA. Without wishing to be bound by theory, 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). The RNase III-like enzyme of Dicer processes the dsRNA to produce short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the invention is a 24-30 nucleotide dsRNA that interacts with the TTR RNA sequence to induce cleavage of the target RNA.

[0119] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. 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 include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide can be present on the 5'-end, the 3'-end, or both ends of either the antisense or sense strand of a dsRNA. In one embodiment of a dsRNA, at least one strand includes a 3'-overhang of at least one nucleotide. In another embodiment, at least one strand comprises 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' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0120] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging, e.g., at the 3' and / or 5' end. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging, e.g., at the 3' and / or 5' end. In another embodiment, one or more nucleotides in the overhang are substituted with a thiophosphate nucleoside.

[0121] In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can comprise an extended length of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In certain embodiments, the extended overhang is present on the sense strand of the duplex. In certain embodiments, the extended overhang is present on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is present on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is present 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 duplex. In certain embodiments, the extended overhang is present on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion such that the overhang is capable of forming a stable hairpin structure under physiological conditions.

[0122] " Blunt-ended " or " blunt-ended " means that there is no unpaired nucleotide at the end of double-stranded RNAi agent, i.e., there is no nucleotide overhang. " Blunt-ended " RNAi agent is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule, dsRNA. The RNAi agent of the present invention includes the RNAi agent that has nucleotide overhang at one end (i.e., the agent that has one overhang and one blunt end) or the RNAi agent that has nucleotide overhang at both ends.

[0123] 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. 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. When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the internal or terminal regions of the molecule. Generally, mismatches are most tolerated within the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends of the iRNA. In one embodiment, a double-stranded RNAi agent of the present invention includes a nucleotide mismatch within the antisense strand. In another embodiment, a double-stranded RNAi agent of the present invention includes a nucleotide mismatch within the sense strand. In one embodiment, the nucleotide mismatch is located, e.g., within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is located, e.g., within the 3'-terminal nucleotide of the iRNA.

[0124] The terms "sense strand" or "passenger strand," as used herein, refer to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as defined herein.

[0125] As used herein, the term "cleavage region" refers to the region that is immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage site is specifically located at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

[0126] 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 the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions to form a double-stranded structure, as would be understood by one of skill in the art. Such conditions can be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, can be applied. One of skill in the art can determine the optimal set of conditions for testing the complementarity of two sequences depending on the end use of the hybridized nucleotides.

[0127] For example, a complementary sequence in an iRNA, such as a dsRNA described herein, includes base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs upon hybridization of a double strand of up to 30 base pairs, while retaining the ability to hybridize under conditions most appropriate for their end use, such as inhibiting gene expression through the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in 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, would still be referred to as "fully complementary" for purposes described herein.

[0128] "Complementary" sequences, as used herein, also include or may be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs.

[0129] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context in which they are used.

[0130] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of a subject mRNA (e.g., an mRNA encoding the TTR gene). For example, a polynucleotide is complementary to at least a portion of a TTR mRNA if its sequence is substantially complementary to a non-interrupted portion of the mRNA encoding the TTR gene.

[0131] 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 fully complementary to SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'). In one embodiment, the antisense polynucleotide sequence is 5'-UCUUGGUUACAUGAAAUCCCAUC-3' (SEQ ID NO: 3).

[0132] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target TTR sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the corresponding region of the nucleotide sequence of any one of SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3'), or a fragment of any one of SEQ ID NOs: 1, 2, and 5, over its entire length.

[0133] In one embodiment, an RNAi agent of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide (which in turn is complementary to a target TTR sequence), wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to a corresponding region of the nucleotide sequence of any one of the sequences in Tables 1, 3, 5, 6, and 7.

[0134] In another embodiment, an RNAi agent of the present invention comprises an antisense strand that is substantially complementary to a target TTR sequence and comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to a corresponding region of the nucleotide sequence of any one of the sequences in Tables 1 and 3 over its entire length.

[0135] In some embodiments, generally, the majority of nucleotides in each strand are ribonucleotides, although one or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides, as described in detail herein. Furthermore, "iRNA" includes ribonucleotides with chemical modifications. Such modifications include all types of modifications disclosed herein or known in the art. For purposes of this specification and claims, all such modifications are encompassed by "iRNA" as used in reference to iRNA molecules.

[0136] In one embodiment of the present invention, the agent used in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner 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 RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0137] As used herein, the term "TTR-related disease" is intended to include any disease associated with the TTR gene or protein. Such diseases may be caused, for example, by excessive production of TTR protein, by TTR gene mutations, by abnormal cleavage of TTR protein, or by abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. "TTR-related disease" includes any type of TTR amyloidosis (ATTR) in which TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposition. 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, and hyperthyroxinemia. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, 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, substantially reduced mBMI (modified body mass index), cranial neuropathy, and lattice corneal dystrophy.

[0138] II. iRNAs of the Invention The present invention provides iRNAs that selectively inhibit the expression of one or more TTR genes. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits TTR gene expression in a cell, such as a cell in a subject, such as a mammal, such as a human, with a TTR-related disease. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during TTR gene expression. The complementary region is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the TTR gene, the iRNA selectively inhibits the expression of the TTR gene (e.g., human, primate, non-primate, or avian Sertpincl gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0139] dsRNA comprises two complementary RNA strands, which hybridize to form a double-stranded structure under the conditions in which dsRNA is used.One strand of dsRNA (antisense strand) comprises a complementary region, which is substantially complementary to the target sequence, and is generally completely complementary.The target sequence can be derived from the sequence of mRNA formed during the expression of TTR gene.The other strand (sense strand) comprises a region complementary to the antisense strand, so that when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0140] Generally, the double-stranded structure is, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 , 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the recited ranges and lengths are also contemplated as part of the invention.

[0141] Similarly, the complementary region of the target sequence may be, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 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 and 15-30 nucleotides in length, such as 7, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the listed ranges and lengths are also contemplated as part of the invention.

[0142] In some embodiments, the dsRNA is about 15 to about 20 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 serve as substrates for Dicer. As those skilled in the art will recognize, the target region of an RNA targeted for cleavage is most often a portion of a larger RNA molecule, which is often an mRNA molecule. Where applicable, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0143] Those skilled in the art will appreciate that the amount of hydroxybenzoates may be, for example, about 10 to 36, 11 to 36, 12 to 36, 13 to 36, 14 to 36, 15 to 36, 9 to 35, 10 to 35, 11 to 35, 12 to 35, 13 to 35, 14 to 35, 15 to 35, 9 to 34, 10 to 34, 11 to 34, 12 to 34, 13 to 34, 14 to 34, 15 to 34, 9 to 33, 10 to 33, 11 to 33, 12 to 33, 13 to 33, 14 to 3 3, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-1 9, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20 It will also be recognized that a double-stranded region, such as a double-stranded region of approximately 9-36 base pairs, such as 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, is the primary functional portion of a dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs is a dsRNA, within the range that it is processed into, for example, a 15-30 base pair functional duplex that targets a desired RNA for cleavage. Thus, one 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 expression is not generated in the target cell by cleavage of a larger dsRNA.

[0144] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides.Compared to their blunt-end counterparts, dsRNAs with at least one nucleotide overhang can have surprisingly superior inhibitory properties.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides.The overhang can be on the sense strand, the antisense strand, or any combination thereof.Furthermore, the overhanging nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense or sense strand of dsRNA.In certain embodiments, longer, extended overhangs are possible.

[0145] dsRNA can be synthesized by standard methods known in the art using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0146] The iRNA compounds of the present invention can be prepared using a two-step method.First, the individual strands of the double-stranded RNA molecule are prepared separately.Then, the component strands are annealed.The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis offers the advantage that it can easily prepare oligonucleotide strands containing unnatural or modified nucleotides.The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0147] 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 1, 3, 5, 6, and 7, and the antisense strand corresponding to the sense strand is selected from the sequences provided in any one of Tables 1, 3, 5, 6, and 7. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the mRNA sequence generated during TTR gene expression. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide being described as the sense strand of any one of Tables 1, 3, 5, 6, and 7, and the second oligonucleotide being described as the antisense strand corresponding to the sense strand of any one of Tables 1, 3, 5, 6, and 7. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0148] Although some of the sequences in Tables 1, 3, 5, 6, and 7 are described as modified and / or conjugated sequences, it is understood that the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, may comprise any one of the sequences set forth in Tables 1, 3, 5, 6, and 7 unmodified, unconjugated, and / or modified and / or conjugated differently than described.

[0149] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of approximately 20-23 base pairs, such as 21 base pairs, have been proposed as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures can also be similarly effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of the oligonucleotide sequences provided in any one of Tables 1, 3, 5, 6, and 7, the dsRNAs described herein may contain at least one strand that is at least 21 nucleotides long. It can be reasonably expected that shorter duplexes having one of the sequences in any one of Tables 1, 3, 5, 6, and 7, missing only a few nucleotides at one or both termini, may be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences in any one of Tables 1, 3, 5, 6, and 7, and whose ability to inhibit TTR gene expression differs by about 5, 10, 15, 20, 25, or 30% or less from a dsRNA containing the full-length sequence, are intended to be within the scope of the present invention.

[0150] Furthermore, the RNAs provided in any one of Tables 1, 3, 5, 6, and 7 identify sites in the TTR transcript that are highly susceptible to RISC-mediated cleavage. Accordingly, the present invention further features iRNAs that target within one of these sequences. As used herein, an iRNA is said to target a specific site in an RNA transcript if it promotes cleavage of the transcript anywhere within that specific site. Such iRNAs generally contain approximately 15 contiguous nucleotides from one of the sequences provided in any one of Tables 1, 3, 5, 6, and 7, linked to additional nucleotide sequences from regions adjacent to the selected sequence in the TTR gene.

[0151] Target sequences are generally about 15-30 nucleotides in length, although there is wide variability in the suitability of specific sequences within this range to induce cleavage of any given target RNA. While the various software packages and guidelines presented herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken, in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed, either physically or figuratively (e.g., by computer simulation), on the target RNA sequence to identify sequences within a size range that can serve as target sequences. By successively moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, while the sequences identified in, for example, any one of Tables 1, 3, 5, 6, and 7 represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by successively "window walking" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0152] It is contemplated that further optimization of any sequence identified in, for example, any one of Tables 1, 3, 5, 6, and 7 can be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking through windows of a size longer or shorter than the target RNA from that position. Again, combining this approach of creating new target candidates with testing the effectiveness of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein can lead to further improvements in inhibition efficiency. Still further, such optimized sequences can be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or modifying overhangs, or other modifications known in the art and / or discussed herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, enhancing transmembrane delivery, targeting specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0153] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the range of mismatches is not located in the center of the complementary region. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA agent strand complementary to a TTR gene region, the RNA strand generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an iRNA containing mismatches with the target sequence is effective in inhibiting TTR gene expression. Examining the effectiveness of an iRNA with mismatches in inhibiting TTR gene expression is important, especially when a specific complementary region of the TTR gene is known to have polymorphic sequence variation within the population.

[0154] III. Modified iRNAs of the Invention In one embodiment, the RNA of an iRNA of the invention, e.g., dsRNA, is native and does not contain chemical modifications and / or linkages, e.g., those known in the art and described herein. In another embodiment, the RNA of an iRNA of the invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. In some embodiments, substantially all of the nucleotides of an iRNA of the invention are modified, and the iRNA contains no more than eight 2'-fluoro modifications on the sense strand (e.g., no more than seven 2'-fluoro modifications, no more than six 2'-fluoro modifications, no more than five 2'-fluoro modifications, no more than four 2'-fluoro modifications, no more than three 2'-fluoro modifications, or no more than two 2'-fluoro modifications) and no more than six 2'-fluoro modifications on the antisense strand (e.g., no more than five 2'-fluoro modifications, no more than four 2'-fluoro modifications, no more than three 2'-fluoro modifications, or no more than two 2'-fluoro modifications). In other embodiments, all of the nucleotides of an iRNA of the invention are modified, and the iRNA contains no more than eight 2'-fluoro modifications on the sense strand (e.g., no more than seven 2'-fluoro modifications, no more than six 2'-fluoro modifications, no more than five 2'-fluoro modifications, no more than four 2'-fluoro modifications, no more than three 2'-fluoro modifications, or no more than two 2'-fluoro modifications) and no more than six 2'-fluoro modifications on the antisense strand (e.g., no more than five 2'-fluoro modifications, no more than four 2'-fluoro modifications, no more than three 2'-fluoro modifications, or no more than two 2'-fluoro modifications). An iRNA of the invention in which "substantially all of the nucleotides are modified" may be extensively, but not entirely, modified and contain no more than five, four, three, two, or one unmodified nucleotides.

[0155] Nucleic acids featured in the present invention can be synthesized and / or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. For example, modifications include terminal modifications, such as 5'-end modifications (phosphorylation, conjugated linkage, inverted linkage) or 3'-end modifications (conjugated linkage, DNA nucleotide, inverted linkage, etc.); base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and backbone modifications, including modification or replacement 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 RNAs that do not contain natural internucleoside linkages. The RNA with modified backbone particularly includes those that do not have phosphorus atom in backbone.For the purpose of this specification, and as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in their internucleoside backbone is also considered to be oligonucleoside.In some embodiments, modified iRNA has phosphorus atom in its internucleoside backbone.

[0156] 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 boranophosphates with reverse polarity, in which adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0157] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; and 5,286,717, each of which is hereby incorporated by reference in its entirety. No. 17; U.S. Patent No. 5,321,131; U.S. Patent No. 5,399,676; U.S. Patent No. 5,405,939; U.S. Patent No. 5,453,496; U.S. Patent No. 5,455,233; U.S. Patent No. 5,466,677; U.S. Patent No. 5,476,925; U.S. Patent No. 5,519,126; U.S. Patent No. 5,536,821; U.S. Patent No. 5,541,316; U.S. Patent No. 5,550,111; U.S. Patent No. 5,563,253; U.S. Patent No. 5,57 Nos. 1,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6 ,531,590; U.S. Patent No. 6,534,639; U.S. Patent No. 6,608,035; U.S. Patent No. 6,683,167; U.S. Patent No. 6,858,715; U.S. Patent No. 6,867,294; U.S. Patent No. 6,878,805; U.S. Patent No. 7,015,315; U.S. Patent No. 7,041,816; U.S. Patent No. 7,273,933; U.S. Patent No. 7,321,029; and U.S. Patent No. RE39464, but are not limited thereto.

[0158] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, those with amide backbones, and others with mixed N, O, S, and CH2 components.

[0159] Representative United States patents that teach the preparation of the above oligonucleosides include U.S. Pat. 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; and 5,470,967, each of which is hereby incorporated by reference in its entirety. 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.

[0160] In another embodiment, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide units, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has 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 are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the contents of each of which are incorporated herein by reference in their entirety. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0161] Some embodiments featured in the present invention include RNAs with phosphorothioate backbones, and oligonucleosides with heteroatom backbones that are --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene(methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (natural phosphodiester backbones are represented as --O--P--O--CH2--) of the aforementioned U.S. Pat. No. 5,489,677, and amide backbones of the aforementioned U.S. Pat. No. 5,602,240. In some embodiments, the RNA featured herein has a morpholino backbone structure as described in the aforementioned US Pat. No. 5,034,506.

[0162] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs, such as dsRNAs provided herein, can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl, or C2-C 10 It can be alkenyl and 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 another embodiment, the dsRNA includes one of the following at the 2' position: C1 to C 10

[0033] In some embodiments, the modification is 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 1996). 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, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described in the Examples herein below.

[0163] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, specifically at the 3' position of the sugar on the 3'-terminal nucleotide, or in 2'-5'-linked dsRNA, and at the 5' position of the 5'-terminal nucleotide. An iRNA can also have a sugar mimic, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of the above modified sugar structures include, certain of which are commonly owned with the present application: U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; and U.S. Pat.

[0010] Examples of patents that may be used include, but are not limited to, U.S. Patent Nos. 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 contents of each of which are hereby incorporated by reference in their entirety.

[0164] The RNA of the iRNA of the present invention may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymine (dT), 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil) ); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, specifically 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 other synthetic and natural nucleobases such as 3-deazaguanine and 3-deazaadenine.Further, the nucleobase can include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by 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 O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0165] Representative United States patents that teach the preparation of the above-mentioned specific modified nucleobases, as well as other modified nucleobases, include the above-mentioned U.S. Pat. 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; and 5,587,469, each of which is hereby incorporated by reference in its entirety. Nos.; U.S. Patent Nos. 5,594,121, 5,596,091; U.S. Patent No. 5,614,617; U.S. Patent No. 5,681,941; U.S. Patent No. 5,750,692; U.S. Patent No. 6,015,886; U.S. Patent No. 6,147,200; U.S. Patent No. 6,166,197; U.S. Patent No. 6,222,025; U.S. Patent No. 6,235,887; U.S. Patent No. 6,380,368; U.S. Patent No. 6,528,640; U.S. Patent No. 6,639,062; U.S. Patent No. 6,617,438; U.S. Patent No. 7,045,610; U.S. Patent No. 7,427,672; and U.S. Patent No. 7,495,088.

[0166] The RNA of an iRNA can also be modified to contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, an agent of the present invention may contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety such that the ribose moiety contains an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce non-specific effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides used in the polynucleotide of the present invention include, but are not limited to, nucleosides that comprise a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides that comprise a bridge from 4' to 2'.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 "hindered 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; see, e.g., U.S. Pat. No. 8,399,845). ,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' (wherein R is H, C1-C12 alkyl, or a 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 hereby incorporated by reference.

[0167] Additional representative U.S. patents and published U.S. patent applications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: 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; and 7,399,845. Nos. 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 hereby incorporated by reference herein.

[0168] Any of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0169] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as "S-cEt."

[0170] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen optimally for stability and affinity, resulting in reduced puckering of the ribose ring.

[0171] 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 hereby incorporated by reference herein.

[0172] One or more of the nucleotides of the iRNA of the invention may also comprise a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide (also referred to as a "non-locked nucleic acid" ("UNA") modification).

[0173] 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 hereby incorporated by reference into this specification).

[0174] Potential stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, and inverted base dT (idT). Disclosure of this modification is found in WO 2011 / 005861.

[0175] Other modifications of the nucleotides of the iRNAs of the invention include a 5' phosphate or 5' phosphate mimic, such as a 5' terminal phosphate or phosphate mimic, on the antisense strand of the RNAi agent. 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.

[0176] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the invention, the double-stranded RNAi agents of the invention comprise chemical modifications, e.g., as disclosed in U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, or International Application No. PCT / US2012 / 065691, filed November 16, 2012, the entire contents of each of which are incorporated herein by reference.

[0177] More specifically, it has been surprisingly discovered that when the sense and antisense strands of a double-stranded RNAi agent are 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 RNAi agent, the gene silencing activity of the RNAi agent is significantly enhanced.

[0178] Thus, the present invention provides double-stranded RNAi 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 may be in the range of 12 to 30 nucleotides in length. For example, each strand may be between 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0179] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA") as a duplex, also referred to herein as an "RNAi agent." The double-stranded region of an RNAi agent may be 12 to 30 nucleotide pairs in length. For example, the double-stranded region may be 14 to 30 nucleotide pairs, 17 to 30 nucleotide pairs, 27 to 30 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0180] In one embodiment, the RNAi agent may include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs can form mismatches with the target mRNA, be complementary to the targeted gene sequence, or be another sequence. The first and second strands can also be linked by additional bases or other non-basic linkers, for example, to form a hairpin.

[0181] In one embodiment, each nucleotide in the overhang region of RNAi agent can be independently modified or unmodified nucleotide, for example, but not limited to, 2'-sugar modified, 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.For example, TT can be the overhang sequence at either end on either strand.The overhang can form a mismatch with target mRNA, or can be complementary to the gene sequence being targeted, or can be another sequence.

[0182] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated.In some embodiments, the overhang region comprises two nucleotides with phosphorothioate between them, wherein the two nucleotides can be the same or different.In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands.In one embodiment, the 3'-overhang is present in the antisense strand.In one embodiment, the 3'-overhang is present in the sense strand.

[0183] RNAi agent may only comprise a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang may be located at the 3' end of the sense strand, or alternatively at the 3' end of the antisense strand.RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without wishing to be bound by theory, it is preferable that the asymmetric blunt end at the 5' end of the antisense strand and the overhang at the 3' end of the antisense strand load the guide strand into RISC process.

[0184] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the RNAi agent is blunt, while the other end comprises a two-nucleotide overhang.Preferably, the two-nucleotide overhang is present at the 3' end of the antisense strand.

[0185] When a two-nucleotide overhang exists at the 3'-end of the antisense strand, two phosphorothioate internucleotide bonds can exist between the three nucleotide ends, where two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.In one embodiment, the RNAi agent further has two phosphorothioate internucleotide bonds between the three nucleotide ends at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, all nucleotides in the sense strand and antisense strand of the RNAi agent, including the nucleotide that is part of the motif, are modified nucleotides.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. In one embodiment, all of the nucleotides of an iRNA of the invention are modified, and the iRNA comprises no more than eight 2'-fluoro modifications on the sense strand (e.g., no more than seven 2'-fluoro modifications, no more than six 2'-fluoro modifications, no more than five 2'-fluoro modifications, no more than four 2'-fluoro modifications, no more than three 2'-fluoro modifications, or no more than two 2'-fluoro modifications) and no more than six 2'-fluoro modifications on the antisense strand (e.g., no more than five 2'-fluoro modifications, no more than four 2'-fluoro modifications, no more than three 2'-fluoro modifications, or no more than two 2'-fluoro modifications). Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0186] In one embodiment, the sense strand of the RNAi agent comprises at least one motif of three identical modifications over three consecutive nucleotides, where one of the motifs is at the cleavage site in the sense strand.

[0187] In one embodiment, the antisense strand of the RNAi agent also contains at least one motif of three identical modifications over three consecutive nucleotides, where one of the motifs is at or near the cleavage site in the antisense strand.

[0188] In RNAi agents having a double-stranded region 17-23 nucleotides long, the cleavage sites in the antisense strand are typically approximately 10, 11, and 12 from the 5' end. Thus, three identically modified motifs may be present at positions 9, 10, and 11; 10, 11, and 12; 11, 12, and 13; 12, 13, and 14; or 13, 14, and 15 of the antisense strand, where counting begins from the first nucleotide from the 5' end of the antisense strand, or counting begins from the first paired nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage site within the antisense strand may also vary depending on the length of the double-stranded region of the RNAi agent from the 5' end.

[0189] The sense strand of RNAi agent can have at least one motif of three identical modifications on three consecutive nucleotides at or near 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 pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0190] In one embodiment, all nucleotides in the sense strand and antisense strand of RNAi agent, including the nucleotide of the 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 non-linked phosphate oxygen and / or one or more linking phosphate oxygen;Modification of ribose sugar components, for example, the 2' hydroxyl of ribose sugar;Modification or substitution of phosphate moiety with " dephosphorylation " linker;Modification or substitution of natural base;And substitution or modification of ribose-phosphate backbone.

[0191] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at positions that are repeated within the nucleic acid. In some cases, modifications will occur at every target position in the nucleic acid, but in many cases, this is not the case. For example, modifications may occur only at the 3' or 5' terminal position, or only within the terminal region, such as a position on the terminal nucleotide or within the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only within the double-stranded region of the RNA, or only within the single-stranded region of the RNA. For example, phosphorothioate modifications at non-linked O positions may occur only at one or both ends, or only within the terminal region, such as a position on the terminal nucleotide or within the last 2, 3, 4, 5, or 10 nucleotides of the chain, or within double-stranded and single-stranded regions, especially at the ends. The 5' end may be phosphorylated.

[0192] For example, it may be possible to 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' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications known in the art at the 2' position of the ribose sugar, such as deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications, in place of the ribosugar of the nucleobase, and modifications at the phosphate group, such as phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0193] In one embodiment, 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.These 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.

[0194] At least two different modifications are typically present on the sense and antisense strands, and the two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0195] In one embodiment, N a and / or N bincludes an alternating pattern 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 strand. The alternating nucleotides may refer to one every other nucleotide or one every third nucleotide, or similar patterns. For example, if A, B, and C each represent one modification type to a nucleotide, the alternating motif could be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0196] 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 modification type to a nucleotide, the alternation pattern, i.e., the modifications to every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several modification possibilities within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0197] In one embodiment, the RNAi agents of the present invention include a modification pattern in an alternating motif on the sense strand that is altered relative to the modification pattern in an alternating motif on the antisense strand. The modification groups of nucleotides in the sense strand may correspond to different modification groups of nucleotides in the antisense strand, or vice versa. For example, when the sense strand pairs with the antisense strand in a dsRNA duplex, within the double-stranded region, the alternating motif in the sense strand may begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BABABA" from the 5'-3' end of the strand. As another example, within the double-stranded region, the alternating motif in the sense strand may begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from the 5'-3' end of the strand, thereby resulting in a complete or partial change in the modification pattern between the sense and antisense strands.

[0198] In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the initial sense strand that has a variation relative to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the initial antisense strand, i.e., 2'-O-methyl modified nucleotides on the sense strand base pair with 2'-F modified nucleotides on the antisense strand, and vice versa. Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification.

[0199] By introducing one or more motifs of three identical modifications on three consecutive nucleotides on sense strand and / or antisense strand, the original modification pattern existing in sense strand and / or antisense strand is interrupted.By introducing one or more motifs of three identical modifications on three consecutive nucleotides on sense strand and / or antisense strand, this interruption of the modification pattern of sense strand and / or antisense strand can unexpectedly enhance the gene silencing activity of target gene.

[0200] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different from the modification of the motif. For example, a portion of a sequence containing a motif is 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 a and N b may be the same or different modifications. a and / or N b may be present or absent when a wing modification is present.

[0201] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide at any position of the strand in the sense strand, the antisense strand, or both strands. For example, the internucleotide linkage modification may occur on every nucleotide on the sense strand and / or the antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand and / 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 or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have variations relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0202] In one embodiment, the RNAi comprises phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region.For example, the overhang region may comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide bonds between the two nucleotides.Internucleotide bond modification may also be provided to link the overhang nucleotide to the terminal paired nucleotide within the double-stranded region.For example, at least 2, 3, 4, or all of the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there may be an additional phosphorothioate or methylphosphonate internucleotide bond that links the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there may be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides may be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0203] In one embodiment, a two-nucleotide overhang is present at the 3'-end of the antisense strand, and two phosphorothioate internucleotide bonds are present between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent may further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0204] In one embodiment, the RNAi agent contains mismatches with the target, within the duplex, or a combination thereof. Mismatches may be present in the overhang region or within the duplex region. Base pairs may be ranked based on their tendency to promote dissociation or melting (for example, the simplest method is to consider the association or dissociation free energy of a particular pairing based on each pairing, but similar or similar analyses can also be used). In terms of promoting dissociation, A:U is preferred to G:C; G:U is preferred to G:C; and I:C is preferred to G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (as described elsewhere herein), are preferred to canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred to canonical pairings.

[0205] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the double-stranded region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0206] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of 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.

[0207] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX)iN b -YYY-N b -(ZZZ) j -N a -n q 3'(I) (In the formula, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a represents oligonucleotide sequences containing independently 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b represents an oligonucleotide sequence containing independently 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; N b 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) Preferably, YYY are all 2'-F modified nucleotides.

[0208] In one embodiment, N a and / or N b includes alternating pattern modifications.

[0209] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.For example, when the RNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (for example, can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), where counting starts from the first nucleotide from the 5' end, or optionally, counting starts from the first paired nucleotide within the double-stranded region from the 5' end.

[0210] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 5'n p -N a -YYY-Nb -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) It can be represented by:

[0211] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0212] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0213] When the sense strand is represented by formula (Id), each N b represents an oligonucleotide sequence containing independently 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 containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X, Y, and Z can be the same or different from each other.

[0214] 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) It may be represented by:

[0215] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0216] In one embodiment, the antisense strand sequence of the RNAi has the 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) (In the formula, k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' represents oligonucleotide sequences containing independently 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; where N b ' and Y' do not have the same modification; and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It may be represented by:

[0217] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0218] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, where counting begins from the first nucleotide from the 5' end, or optionally, counting begins from the first paired nucleotide within the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0219] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0220] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0221] Thus, the antisense strand has the following formula: 5'n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p '3'(IIb); 5'n q '-N a '-Y'Y'Y'-N b '-X'X'X'-n p '3' (IIc); or 5'n q '-N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p '3' (IId) It can be represented by:

[0222] 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 ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0223] 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 ' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0224] When the antisense strand is represented by formula (IId), each N b Each N' represents an oligonucleotide sequence that independently contains 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. b is 0, 1, 2, 3, 4, 5 or 6.

[0225] 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) It may be represented by:

[0226] When the antisense strand is represented by formula (IIa), each N aX', Y', and Z' 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 or different from one another. Each nucleotide in the sense and antisense strands may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. Each of X, Y, Z, X', Y', and Z' may specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0227] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21 nt, counting starting at the first nucleotide from the 5' end, or optionally counting starting at the first paired nucleotide within the double-stranded region from the 5' end, and Y representing a 2'-F modification.

[0228] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, counting starting from the first nucleotide from the 5' end, or optionally counting starting from the 5' end with the first paired nucleotide within the double-stranded region, and Y' representing a 2'-O-methyl modification.

[0229] Each of the sense strands represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0230] Thus, the RNAi agent used in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex may have the formula (III): Sense:5'n p -Na -(XXX)iN 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) (In the formula, i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' represents oligonucleotide sequences containing independently 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; where each n p ',n p , n q ', and n q each may be present or absent and independently represent an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0231] 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.

[0232] An exemplary combination of sense and antisense strands that form an RNAi duplex is of the following formula: 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 -YYY-N b -ZZZ-N 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 -XXX-N b -YYY-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N 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) 5'-N a -YYY-N b -3' 3'n p '-N a '-Y'Y'Y'-N b '5' (IIIe) Includes:

[0233] When the RNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0234] When the RNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence that independently contains 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0235] When the RNAi agent is represented by formula (IIIc), each N b , N b Each N' represents an oligonucleotide sequence that independently contains 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0236] When the RNAi agent is represented by formula (IIId), each N b , N b ' represents an oligonucleotide sequence that independently contains 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 N' represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b' each independently includes an alternating pattern of modifications.

[0237] When the RNAi agent is represented by formula (IIIe), each N a , N a ', Nb, and N b ' represents an oligonucleotide sequence comprising 0-25 nucleotides that are independently either modified or unmodified or a combination thereof, each sequence comprising at least two differently modified nucleotides.

[0238] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe) may be the same as or different from one another.

[0239] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), at least one of the Y nucleotides may be base-paired with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0240] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide; or all three of the Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0241] When an RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may be base-paired with one of the X' nucleotides, alternatively, at least two of the X nucleotides are base-paired with the corresponding X' nucleotide; or all three of the X nucleotides are base-paired with the corresponding X' nucleotide.

[0242] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and / or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0243] In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, and n is linked to the adjacent nucleotide via a phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker (described below). p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds, and the sense strand contains at least one phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0244] In one embodiment, when the RNAi agent is represented by Formula (IIIa), the Na modification is a 2'-O-methyl or a 2'-fluoro modification, and n p '>0 and at least one n p' is linked to adjacent nucleotides via phosphorothioate bonds, and the sense strand contains at least one phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0245] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe) are linked to each other at one or both of the 5' and 3' ends, and optionally bound to a ligand. Each of the agents can target the same gene or two different genes, or each of the agents can target the same gene at two different target sites.

[0246] Various publications describe multimeric RNAi agents that can be used in the methods of the present invention, including WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520 (the entire contents of each of which are hereby incorporated by reference).

[0247] As described in further detail below, an RNAi agent comprising one or more carbohydrate moieties attached thereto can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with a non-carbohydrate (preferably cyclic) carrier to which another moiety, such as 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 a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0248] The ligand may be attached to the polynucleotide by a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, e.g., a phosphate backbone, or, e.g., a sulfur-containing modified phosphate backbone. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. The moiety may be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally allow for a bond suitable for incorporation or tethering of another chemical entity, such as a ligand, to the constituent ring.

[0249] The RNAi agent may be attached to the ligand via a carrier, where the carrier can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0250] In certain specific embodiments, for example, an RNAi agent used in the methods of the invention is an agent selected from the group of agents listed in any one of Tables 1, 3, 5, 6, and 7. These agents may further comprise a ligand.

[0251] In certain embodiments, the RNAi agent of the present invention is an agent selected from the group consisting of AD-66016, AD-65492, AD-66017, and AD-66018.

[0252] IV. Ligand-conjugated iRNA Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties, or complexes that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, for example, di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0253] In one embodiment, the ligand changes the distribution, targeting or life span of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides improved affinity to a selected target, such as a molecule, a cell or cell type, a compartment, such as a subcellular or organ compartment, a tissue or organ or region of the body, for example, compared to a chemical species in the absence of such a ligand. Preferred ligands do not participate in double-stranded pairing in duplexed nucleic acid.

[0254] Ligands can include natural 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-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-glycolied) 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 are polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or alpha helical peptides.

[0255] The ligand may also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, for example, an antibody that binds to a specific cell type, such as a kidney cell. The targeting group may be thyroid-stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, monovalent galactose, N-acetyl-galactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimic. In certain embodiments, the ligand includes monovalent or polyvalent galactose. In certain embodiments, the ligand includes cholesterol.

[0256] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralene, 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-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithophosphate, and the like. Examples of suitable cleavage inhibitors include acetylcholinesterases (e.g., acetylcholinesterase ...

[0257] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for co-ligands; or antibodies, such as antibodies that bind to specific cell types, such as liver cells. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0258] The ligand can be a substance, such as a drug, that can increase uptake of an iRNA agent into a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cell's cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0259] In some embodiments, the ligand attached to the iRNA described herein refers to a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, such as, for example, about 5-, 10-, 15-, or 20-base oligonucleotides containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0260] Ligand-conjugated oligonucleotides of the invention may be synthesized by using oligonucleotides bearing pendant reactive functional groups, such as those derived from the addition of a binding molecule onto an oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing an attached binding moiety.

[0261] The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely produced through well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including Applied Biosystems (Foster City, Calif.). Additionally or alternatively, any other means for such synthesis known in the art may be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0262] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing a ligand molecule, or building blocks bearing a non-nucleoside ligand.

[0263] When using a nucleotide conjugate precursor that already has a binding moiety, synthesis of the sequence-specific linked nucleoside is typically completed, and then a ligand molecule is reacted with the binding moiety to produce 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 standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0264] A. Lipid Complex In one embodiment, the ligand or complex is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of complex to target tissue, for example, non-renal target tissue of the body.For example, the target tissue can be the liver, including the parenchymal cells of the liver.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the degradation resistance of complex, (b) increase the targeting or transport to target cell or cell membrane, and / or (c) be used to regulate the binding of serum protein, for example, HSA.

[0265] For example, lipid-based ligand can be used for inhibition, such as controlling the binding of complex to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney, and therefore less likely to be removed from the body.The lipid or lipid-based ligand that binds weaker to HSA can be used to target complex to the kidney.

[0266] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed.

[0267] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells may also be used in place of or in addition to the lipid-based ligand.

[0268] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant types, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, e.g., liver cells. Also included are HSA and low-density lipoprotein (LDL).

[0269] B. Cell-penetrating agents In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the cell-penetrating agent is amphipathic. An exemplary cell-penetrating agent is a peptide such as tat or antennopedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidylmimetic, invertomer, non-peptide or pseudo-peptide bond, and D-amino acid use. The helical agent is preferably an α-helical agent with a lipophilic and lipophobic phase.

[0270] 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. The addition of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by facilitating cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0271] 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 crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can transport a number of polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 14)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage-display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). For cell targeting purposes, an example of a peptide or peptidomimetic tethered to a dsRNA agent through an incorporated monomer unit is an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications to increase stability or induce conformational properties. Any of the structural modifications described below can be used.

[0272] The RGD peptide used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues.RGD-containing peptides and peptidomimetics include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties that target integrin ligands can be used.Preferred complexes of this ligand target PECAM-1 or VEGF.

[0273] 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 cecropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two key amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, cell-penetrating peptides can be bisected amphipathic peptides, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0274] C. Carbohydrate Complex In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, "carbohydrate" refers to either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having a carbohydrate moiety as part of its structure, composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of TTR or greater (e.g., TTR, C6, C7, or C8); di- and trisaccharides include sugars with two or three monosaccharide units (e.g., TTR, C6, C7, or C8).

[0275] In one embodiment, the carbohydrate complexes used in the compositions and methods of the present invention are monosaccharides. In another embodiment, the carbohydrate complexes used in the compositions and methods of the present invention are [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0276] In one embodiment, the monosaccharide is N-acetylgalactosamine, e.g., [ka] is.

[0277] Other exemplary carbohydrate complexes for use in the embodiments described herein include, but are not limited to: [ka] (Formula XXIII) (wherein when one of X or Y is an oligonucleotide, the other is hydrogen).

[0278] 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.

[0279] In one embodiment, a double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to an iRNA agent. In another embodiment, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0280] In some embodiments, for example, when the two strands of an iRNA agent of the 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 each strand of the other, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide within the hairpin loop may comprise a GalNAc or GalNAc derivative independently linked via a monovalent linker. Hairpin loops may also be formed by extended overhangs on one strand of the duplex.

[0281] 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 and / or a cell-penetrating peptide.

[0282] Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publications WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0283] D. Linker In some embodiments, the conjugates or ligands described herein may be attached to the iRNA oligonucleotide by various linkers, which may be cleavable or non-cleavable.

[0284] The term "linker" or "linking group" means an organic moiety that joins two parts of a compound, for example, by covalently bonding the two parts of the compound. Linkers are typically 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 group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, and the like, wherein one or more methylenes are selected from O, S, S(O), SO, N(R 8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (wherein R8 is hydrogen, acyl, aliphatic, or substituted aliphatic). In one embodiment, the linker is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-16 atoms, or 8-16 atoms.

[0285] A cleavable tether is one that is sufficiently stable outside the cell but is cleaved upon entry into a target cell to release the two moieties tethered by the linker. In preferred embodiments, the cleavable tether is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold more rapidly in the target cell, or under first standard conditions (e.g., which may be selected to mimic or correspond to intracellular conditions), than in the subject's blood, or under second standard conditions (e.g., which may be selected to mimic or correspond to conditions found in blood or serum).

[0286] Cleavable linking groups are susceptible to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common in cells than in serum or blood, or are found at higher levels or activity. Examples of such degradable agents include oxidizing or reducing enzymes or reducing agents such as mercaptans present in cells, which can degrade redox-cleavable linking groups by reduction, and are selective for specific substrates or do not have substrate specificity; esterases; agents that can create an acidic environment, such as endosomes or those that produce a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate specific), and phosphatases.

[0287] Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. 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 about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand in the cell or to a desired compartment of the cell.

[0288] Linker can contain cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the cell to be targeted.For example, the ligand for targeting liver can be linked to cationic lipid through a linker that contains ester group.Hepatocytes are rich in esterase, therefore linker is more efficiently cleaved 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.

[0289] Linkers containing peptide bonds may be used in targeting peptidase-rich cell types such as hepatocytes and synoviocytes.

[0290] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradable agent (condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, with the first condition selected to indicate cleavage in target cells and the second condition selected to indicate 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 a whole animal. It may be useful to perform initial evaluation in a cell-free or culture condition and confirm with further evaluation in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more rapidly 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).

[0291] i. Redox-cleavable linker In one embodiment, the cleavable linker is a redox-cleavable linker that is cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can rely on the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. A candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly 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 a candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0292] ii. Phosphate-based cleavable linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker. The phosphate-based cleavable linker can be cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves phosphate groups in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0293] iii. Acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment of about pH 6.5 or below (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or below) or by an agent such as an enzyme that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when a carbon is attached to the oxygen of the ester (alkoxy group), the group is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates may be evaluated using methods similar to those described above.

[0294] iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved intracellularly by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0295] v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give rise to 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 alkynelene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, but do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates may be evaluated using methods similar to those described above.

[0296] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrates conjugated to linkers in the compositions and methods of the present invention include: [ka] [ka] (In the formula, wherein one of X or Y is an oligonucleotide and the other is hydrogen).

[0297] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

[0298] In one embodiment, the dsRNA of the invention is Formulas (XXXII) to (XXXV), [ka] (In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent each occurrence from 0 to 20, and q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent each occurrence from 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 5C is, each independently for 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 for each occurrence absent, alkylene, or substituted alkylene, and 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 is independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., independently for each occurrence, a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain). The trivalent conjugated GalNAc derivative is conjugated to a bivalent or trivalent branched linker selected from the group of structures represented by: Formula (XXXVI), [ka] (In the formula, L 5A , L 5B and L 5C It is particularly useful for use with RNAi agents to inhibit the expression of target genes such as ribonucleotides (wherein represents a monosaccharide, such as a GalNAc derivative).

[0299] Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas II, VII, XI, X, and XIII.

[0300] Representative United States patents that teach the preparation of RNA complexes include U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; and 5,591,584, the contents of each of which are hereby incorporated by reference in their entirety. Details; U.S. Patent Nos. 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737 Nos.; U.S. Patent Nos. 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469 ;U.S. Patent No. 5,258,506;U.S. Patent No. 5,262,536;U.S. Patent No. 5,272,250;U.S. Patent No. 5,292,873;U.S. Patent No. 5,317,098;U.S. Patent No. 5,371,241, U.S. Patent No. 5,391,723;U.S. Patent No. 5,416,203, U.S. Patent No. 5,451,463;U.S. Patent No. 5,510,475;U.S. Patent No. 5,512,667;U.S. Patent No. 5,514,785;U.S. Patent No. 5,565,552;Examples of such patents include, but are not limited to, U.S. Patent Nos. 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 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.

[0301] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the foregoing modifications may be incorporated in a single compound, or even in a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0302] "Chimeric" iRNA compounds or "chimeras," in the context of the present invention, are iRNA compounds, preferably dsRNA, that contain two or more chemically distinct regions, each composed of at least one monomer unit, i.e., nucleotides in the case of dsRNA compounds. These iRNAs typically contain at least one region in which the RNA has been modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / 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. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H therefore results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. As a result, comparable results are often obtained with shorter iRNAs when chimeric dsRNAs are used compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0303] In some cases, the RNA of an iRNA may be modified by a non-ligand group. To enhance the activity, cellular distribution, or intracellular uptake of an iRNA, several non-ligand molecules have been conjugated to the iRNA, and procedures for performing such conjugation 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), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents that teach the preparation of such RNA complexes 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 to be conjugated using an appropriate coupling or activation reagent. The conjugation reaction can be carried out in solution phase while the RNA is still bound to the solid support, or following RNA cleavage. Purification of the RNA complex by HPLC typically yields a pure complex.

[0304] V. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, such as a cell in a subject, e.g., a human subject (e.g., a subject in need thereof, e.g., a subject with a TTR-related disease, disorder, or condition), can be achieved in several different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention, either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition containing an iRNA, e.g., dsRNA, to the subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and induce expression of the iRNA. These alternatives are discussed further below.

[0305] 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, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. WO 94 / 02595, the entire contents of which are incorporated herein by reference). 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. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or implantation into tissue or local administration of the formulation. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure to the agent in systemic tissues that may otherwise be harmed by or degrade the agent, and allows for administration of a lower total dose of the iRNA molecule. Several studies have demonstrated successful gene product knockdown when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and by subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) has been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and prolonged the survival of tumor-bearing mice (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523).RNA interference can be delivered to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lung by intranasal administration (Howard, KA., et (2006) Mol. Ther. 14:476-484; Zhang, X., et al. (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al. (2005) Nat. Med. 11:50-55) have demonstrated successful localized delivery. To administer iRNA systemically to treat disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent rapid degradation of dsRNA by endogenous endo- and exonucleases. Modification of the RNA or pharmaceutical carrier can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical attachment of lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, systemic injection of iRNAs directed against ApoB conjugated to lipophilic cholesterol moieties into mice resulted in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J., et al. (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to suppress tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, J.O., et al. (2006) Nat. Biotechnol. 24:1005-1015).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 facilitate the binding of iRNA molecules (which are negatively charged) and also enhance their interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNAs or induced to form vesicles or micelles that encapsulate iRNAs (see, for example, Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNAs upon systemic administration. Methods for making and administering cationic iRNA complexes are well within the capabilities of one of ordinary skill in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N., et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al. (2007) J. Hypertens. 25:197-205, the contents of which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N., et al. (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S., et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y., et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E., et al. (2005) Int J. Oncol. 26:1087-1091), and PEG-1 (Polymerase Chain Receptor Blockers). al (2008) Pharm. Res. August 16, advance online publication; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, for systemic administration, the iRNA is complexed with cyclodextrin. Methods and pharmaceutical compositions for administering iRNA and cyclodextrin are described in U.S. Patent No. 7,427,605, the entire contents of which are incorporated herein by reference.

[0306] A. Vectors Encoding iRNAs of the Invention TTR gene-targeting iRNAs 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., WO 00 / 22113; Conrad, WO 00 / 22114; and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (hours to weeks) or persistent (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0307] Each iRNA strand or strands can be transcribed from the promoter on the expression vector.When expressing two separate strands to produce, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or infection).Alternatively, each of the promoters can be transcribed from the individual strands of dsRNA by being located on the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat polynucleotide that is linked by a linker polynucleotide sequence, so that dsRNA has a stem-loop structure.

[0308] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors can be by systemic administration, such as intravenous or intramuscular administration, administration to target cells explanted from a patient and then reintroduced into the patient, or any other means that allows for introduction into desired target cells.

[0309] iRNA expression plasmids can be transfected into target cells as complexes with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown, targeting different regions of a target RNA over a period of one week or more, are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells in vitro can be ensured using markers that confer resistance to specific environmental factors (e.g., antibiotics and drugs) on transfected cells, such as hygromycin B resistance.

[0310] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may contain viral sequences for transfection, if desired. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure iRNA expression in target cells. Other contemplated aspects of vectors and constructs are described in more detail below.

[0311] Vectors useful for delivering iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulated / inducible expression.

[0312] The expression of iRNA can be precisely regulated using inducible regulatory sequences that are sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24).Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG).Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0313] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to patients. More details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells, for example, to generate stem cells that are more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors that may be used include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; U.S. Patent No. 5,665,557; and U.S. Patent No. 5,981,276, which are incorporated herein by reference.

[0314] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0315] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNAs can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, e.g., with either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101; Fisher KJ et al. (1996), J.Virol, 70:520-532; Samulski R et al. (1989), J.Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; WO 94 / 13788; and WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0316] Another viral vector suitable for delivering the iRNA of the invention is a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or a poxvirus, e.g., an avipox, e.g., fowlpox or canarypox.

[0317] The tropism of viral vectors can be modified, if necessary, by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by expressing different capsid protein serotypes; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0318] The vector pharmaceutical preparation can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0319] VI. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNAs of the invention. In one embodiment, the present invention provides a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with TTR gene expression or activity. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration through parenteral delivery, e.g., by subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma, e.g., by intracerebral infusion, such as continuous pump infusion. Pharmaceutical compositions of the invention may be administered at a dose sufficient to inhibit TTR gene expression. In one embodiment, an iRNA agent, e.g., a dsRNA agent, of the invention is formulated for subcutaneous administration in a pharmaceutically acceptable carrier.

[0320] The pharmaceutical composition can be administered by intravenous infusion over a period of time, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about 25 minutes. Administration can be repeated periodically, for example, weekly or every other week (i.e., every two weeks), for one month, two months, three months, four months, or longer. Administration can also be repeated, for example, monthly or quarterly, for example, about every 12 weeks. After the initial treatment regimen, the therapeutic agent can be administered less frequently. For example, after three months of weekly or biweekly administration, administration can be repeated monthly for six months or a year or more.

[0321] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more subdoses at appropriate intervals throughout the day, or even via continuous infusion or controlled-release delivery. In this case, the amount of iRNA contained in each subdose must be correspondingly smaller to achieve the total daily dose. The dosage unit can also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of the iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for site-specific agent delivery, such as those used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0322] In another embodiment, a single dose of the pharmaceutical composition can be administered over an extended period of time, such that subsequent doses are administered no more than 3, 4, or 5 days apart, no more than 1, 2, 3, or 4 weeks apart, or no more than 9, 10, 11, or 12 weeks apart. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered twice a month. In other embodiments, a single dose of the pharmaceutical composition of the present invention is administered monthly. In yet other embodiments, a single dose of the pharmaceutical composition of the present invention is administered quarterly.

[0323] Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other diseases present, may influence the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. The effective dosage and in vivo half-life of the individual iRNAs encompassed by the present invention may be estimated using conventional procedures or based on in vivo studies using appropriate animal models, as described elsewhere herein.

[0324] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary, for example, by inhalation or insufflation of powders or aerosols, including nebulizers; intratracheal, intranasal, transepidermal, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal administration, for example, via an implanted device; or intracranial administration, for example, intracerebral parenchyma, intrathecal, or intraventricular.

[0325] The iRNA can be delivered in a manner that targets a specific tissue, such as the liver (e.g., liver parenchymal cells).

[0326] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, 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 iRNA featured in the present invention is in admixture with a topical delivery agent, such as a lipid, liposome, fatty acid, fatty acid ester, steroid, chelating agent, or surfactant. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA featured in the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNA can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Topical formulations include, but are not limited to, alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0327] A. iRNA formulations containing membrane-like molecular assemblies The iRNAs used in the compositions and methods of the present invention may be formulated for delivery within membrane-like molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar or multilamellar vesicles, with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition, but may in some cases. Liposomes are useful for transporting and delivering active ingredients to their site of action. Because the liposome membrane is structurally similar to biological membranes, upon application of the liposome to a tissue, the liposomal bilayer fuses with the bilayer of the cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the iRNA, are delivered into the cell, where the iRNA can specifically bind to the target RNA and mediate iRNA activity. In some cases, liposomes are also specifically targeted, for example, to direct iRNA to a particular cell type.

[0328] Liposomes containing iRNA agents can be prepared by a variety of methods. In one example, the lipid components of the liposomes are dissolved in a detergent so that micelles form without the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid complexes. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the iRNA agent, condensing around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to yield a liposome preparation of the iRNA agent.

[0329] If necessary, a carrier compound, e.g., to aid in condensation, can be added during the condensation reaction by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to aid in condensation.

[0330] Methods for generating stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as structural components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. al.Biochim.Biophys.Acta 557:9,1979;Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978;Mayhew,et al.Biochim.Biophys.Acta 775:169,1984;Kim,et al.Biochim.Biophys.Acta 728:339,1983; and Fukunaga, et al. The present invention may also include one or more embodiments of the exemplary methods described in [Endocrinol. 115:757, 1984]. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and a combination of freeze-thawing and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50-200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to the packaging of iRNA agent preparations within liposomes.

[0331] Liposomes are divided into two broad classes: cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes; the positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized inside endosomes; the acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0332] pH-sensitive or negatively charged liposomes do not complex with nucleic acids but rather encapsulate them. Because both nucleic acids and lipids have similar charges, repulsion occurs rather than complexation. Nevertheless, some nucleic acid is encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected within the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0333] One major type of liposome composition contains phospholipids in addition to naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0334] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0335] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their efficacy in delivering drugs to the skin. Cyclosporine A was delivered into the dermis of mouse skin using nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results suggested that such nonionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0336] Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the improved circulation half-life of these sterically stabilized liposomes is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0337] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) describe the use of monosialoganglioside G M1 reported the ability of galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G M1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0338] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot fuse efficiently with the plasma membrane, but can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0339] Additional advantages of liposomes include: Liposomes derived from natural phospholipids are biocompatible and biodegradable; Liposomes can incorporate a wide range of water- and lipid-soluble drugs; and Liposomes can protect iRNA agents encapsulated in their internal compartments from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0340] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the plasma membrane of tissue culture cells, resulting in iRNA agent delivery (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355, for a description of DOTMA and its use in combination with DNA).

[0341] The ADOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells. It contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complexes is also positive. The positively charged complexes thus prepared spontaneously adhere to negatively charged cell surfaces and fuse with the plasma membrane, efficiently delivering functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is attached by an ester rather than an ether bond.

[0342] Other reported cationic lipid compounds include those conjugated to a variety of moieties, including carboxyspermine conjugated to one of two lipid types, such as compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0343] Another cationic lipid complex involves derivatizing lipids with cholesterol ("DC-Chol") in combination with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California), and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0344] Liposomal formulations are particularly suitable for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer the iRNA agent intradermally. In some implementations, liposomes are used to deliver iRNA agents to epidermal cells and to enhance the penetration of iRNA agents into skin tissues, such as within the skin. For example, liposomes can be applied topically. Topical delivery of therapeutic agents formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R M and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0345] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their utility in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0346] Liposomes containing iRNA can be highly deformable. Such deformability can allow the liposome to penetrate pores smaller than the average diameter of the liposome. For example, transferosomes are a type of deformable liposome. Transferosomes can be created by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing iRNA agents can be delivered subcutaneously, for example, by infection, to deliver the iRNA agent to keratinocytes within the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. Furthermore, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and are often self-loading.

[0347] Other formulations consistent with the present invention are described in PCT Publication No. WO 2008 / 042973, the entire contents of which are incorporated herein by reference.

[0348] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to the shape of skin pores), self-repair, often reach their targets without fragmentation, and are often self-loading. To create transfersomes, a surface edge activator, usually a surfactant, can be added to a standard liposome composition. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0349] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and ranking the properties of the many different surfactant types, both natural and synthetic, is by using the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0350] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used over a wide pH range. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0351] If surfactant molecule carries negative charge when dissolved or dispersed in water, surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylate, acyl amide of amino acid, sulfate esters such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate.The most important members of anionic surfactant class are alkyl sulfate and soap.

[0352] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0353] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

[0354] The use of surfactants in pharmaceutical preparations and emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0355] The iRNA used in the methods of the present invention can also be provided as a micellar formulation. A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure so that the hydrophobic portions of the molecules all face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. If the environment is hydrophobic, the opposite arrangement will exist.

[0356] Mixed micelle formulations suitable for transdermal delivery include siRNA compositions, alkali metal C8-C 22The mixed micelle may be prepared by mixing an aqueous solution of alkyl sulfate and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, hyaluronic acid pharmaceutically acceptable salts, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polidocanol alkyl ether and its analogs, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles can be formed regardless of the mixing of the components, but to provide smaller micelles, vigorously mixing is required.

[0357] In one method, a first micelle composition is prepared, containing an siRNA composition and at least an alkali metal alkyl sulfate.The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition.In another method, a micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by adding the remaining micelle-forming compounds with vigorous mixing.

[0358] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added along with the micelle-forming components. An isotonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0359] To deliver a micelle formulation as a spray, the formulation can be placed in an aerosol dispensing device, which can then be loaded with the propellant. The propellant, under pressure, is in liquid form within the dispensing device. The ratio of the components is adjusted so that the aqueous phase and the propellant phase are one, i.e., one phase. If there are two phases, the dispensing device must be shaken before dispensing a portion of the contents, for example, through a metered valve. The dispensed dose of the pharmaceutical product is expelled from the metered valve in a fine spray.

[0360] Propellants include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0361] The specific concentrations of the essential ingredients can be determined by relatively simple experimentation. For absorption through the oral cavity, it is often desirable to increase the dose, e.g., at least two or three times, that for administration through injection or through the gastrointestinal tract.

[0362] B. Lipid particles For example, an iRNA, such as a dsRNA, of the present invention may be formulated fully encapsulated in a lipid formulation, such as, for example, an LNP or other nucleic acid-lipid particle.

[0363] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs exhibit long circulatory life following intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the site of administration), making them extremely useful for systemic applications. LNPs include "pSPLPs" containing encapsulated condensing agent-nucleic acid conjugates, as described in International Publication No. WO 00 / 03683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Additionally, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles, and methods for preparing them, are disclosed, for example, in U.S. Pat. No. 5,976,567; U.S. Pat. No. 5,981,501; U.S. Pat. No. 6,534,484; U.S. Pat. No. 6,586,410; U.S. Pat. No. 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and WO 96 / 40964.

[0364] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above-cited ranges are also considered part of the invention.

[0365] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA). 1,2-Dilinoleyl-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or mixtures thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

[0366] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0367] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (molar percentages), with a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0368] Ionic / non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-2-methylpropional (DOPE), and dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-2-methylpropional (DOPE). The lipid may be an anionic or neutral lipid, including, but not limited to, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0369] The conjugated lipid that inhibits particle aggregation can be, for example, without limitation, a polyethylene glycol (PEG)-lipid, including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (Ci8). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particles.

[0370] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0371] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be created using lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, the contents of which are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution can then be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) to achieve a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously when mixed.Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc.).In some cases, the extrusion step can be omitted.Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration.The buffer can be exchanged with phosphate buffered saline (PBS) at about pH 7, for example, about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. [ka]

[0372] LNP01 formulations are described, for example, in WO 2008 / 042973, which is incorporated herein by reference.

[0373] Additional exemplary lipid dsRNA formulations are described in Table 1.

[0374] [Table 1]

[0375] [Table 2]

[0376] [Table 3]

[0377] Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0378] Formulations containing XTC are described in PCT Publication No. WO 2010 / 088537, the entire contents of which are hereby incorporated by reference.

[0379] Formulations containing MC3 are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference.

[0380] Formulations containing ALNY-100 are described in PCT Publication No. WO 2010 / 054406, the entire contents of which are hereby incorporated by reference.

[0381] Formulations containing C12-200 are described in PCT Publication No. WO 2010 / 129709, the entire contents of which are hereby incorporated by reference.

[0382] Compositions and preparations for oral administration include powder or granule, microparticle, nanoparticle, suspension or solution in water or non-aqueous medium, capsule, gel capsule, sachet, tablet or mini-tablet.Thickener, flavoring agent, diluent, emulsifier, dispersing aid or binder may be required.In some embodiments, oral preparations are those in which the DsRNA of the present invention is administered in combination with one or more penetration-promoting surfactants and chelating agents.Suitable surfactants include fatty acid and / or ester or their salt, bile acid and / or their salt. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as fatty acid / salts combined with bile acids / salts. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA featured in the present invention can be orally delivered in granular form, including spray-dried particles, or can be complexed to form micro- or nanoparticles.DsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkylcyanoacrylates, DEAE-derivatized polyimines, pullulans, cellulose, and starch.Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate. acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0383] Compositions and formulations for parenteral, intraparenchymal (intracerebral), intrathecal, intraventricular, or intrahepatic administration can include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0384] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. When treating liver disorders, such as liver cancer, liver-targeted formulations are particularly preferred.

[0385] The pharmaceutical preparation of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include combining active ingredients with pharmaceutical carriers or excipients. Generally, the preparation is prepared by uniformly and intimately combining active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0386] The compositions of the present invention can be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension can also contain stabilizers.

[0387] C. Additional Formulations i. Emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid in the form of droplets, usually greater than 0.1 μm in diameter (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and (See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems containing two immiscible liquid phases intimately mixed and dispersed within one another. In general, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed and dispersed as minute droplets within the bulk oily phase, the resulting composition is referred to as a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed and dispersed as minute droplets within the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o / w) emulsion.In addition to the dispersed phase and the active agent, which may be present as a solution in either the aqueous or oily phase or as a separate phase, emulsions may contain additional components. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions containing more than two phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages not offered by simple binary emulsions. Multiple emulsions in which individual oil droplets of an o / w emulsion surround small water droplets constitute w / o / w emulsions. Similarly, oil droplet systems encapsulated in globules of water and stabilized within an oily continuous phase provide o / w / o emulsions.

[0388] Emulsions are characterized by little or no thermodynamic stability. Frequently, the dispersed or discontinuous phase of an emulsion is well dispersed within the external or continuous phase and is maintained in this form through the use of emulsifiers or formulation viscosity. Either of the emulsion phases can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Another means of stabilizing emulsions involves the use of emulsifiers, which can be incorporated into either of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0389] Synthetic surfactants, also known as surface active agents, have a wide range of uses in emulsion formulations and have been reviewed in literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199).Surfactants are typically amphiphilic, and comprise hydrophilic and hydrophobic parts. The ratio of hydrophilicity to hydrophobicity is called the hydrophilic / lipophilic balance (HLB) of surfactant, and is a useful tool for classifying and selecting surfactant in the preparation of formulation.Surfactant can be classified into different classes based on the nature of hydrophilic group: nonionic, anionic, cationic and amphoteric (see for example Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285).

[0390] Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorbent bases with hydrophilic properties, such as anhydrous lanolin and hydrophilic petrolatum, can absorb water to form water-in-oil emulsions while still maintaining their semi-solid consistency. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, especially in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0391] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion, including fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0392] Hydrophilic colloids, or hydrocolloids, include natural gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around dispersed phase droplets and by increasing the viscosity of the external phase.

[0393] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can easily support the growth of microorganisms, so preservatives are often incorporated into these preparations.The commonly used preservatives contained in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also generally added to emulsion preparations to prevent the preparation from deteriorating.The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, butylated hydroxytoluene; or reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0394] The application of emulsion formulations via dermal, oral, and parenteral routes and methods for preparing them have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of preparation and efficiency in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutrients are among the materials commonly administered orally as o / w emulsions.

[0395] ii. Microemulsions In one embodiment of the present invention, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphile that is a single optically isotropic and thermodynamically stable solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, microemulsions are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a transparent system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are typically prepared through the combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0396] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with comprehensive knowledge of microemulsion formulation (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs into a formulation of spontaneously formed, thermodynamically stable droplets.

[0397] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. Cosurfactants, which are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant film, resulting in irregular coatings due to the gaps between surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, aqueous pharmaceutical solutions, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0398] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential drug absorption enhancement due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides, or iRNAs. Microemulsions have been effective in transdermal delivery of active ingredients for both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improve local cellular uptake of iRNA and nucleic acids.

[0399] The microemulsions of the present invention may also contain additional ingredients and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve the properties of the formulation and enhance the absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0400] iii. Particulates The iRNA agents of the invention may be incorporated into particles, such as, for example, microparticles. Microparticles can be produced by spray drying, but they can also be produced by other methods, including freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0401] iv. Penetration enhancers In one embodiment, the present invention uses various penetration enhancers to achieve efficient delivery of nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, usually, only lipid-soluble or lipophilic drugs can easily pass through cell membranes. It has been discovered that even non-lipophilic drugs can pass through cell membranes if the membrane they pass through is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0402] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the aforementioned classes of penetration enhancers is described in more detail below.

[0403] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved iRNA absorption through mucosal membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0404] Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1~20 These include alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.). (See, for example, Touitou, E., et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J.Pharm.Pharmacol., 1992, 44, 651-654).

[0405] The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE).(See, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0406] Chelating agents used in the context of the present invention can be defined as compounds that complex with metal ions, removing them from solution and resulting in improved iRNA absorption through mucous membranes. Regarding their use as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as deoxyribonuclease inhibitors, since most DNA nucleases require divalent metal ions for catalysis and are inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines). (See, e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0407] As used herein, a non-chelating, non-surfactant penetration enhancer may be defined as a compound that demonstrates insignificant activity as a chelating agent or as a surfactant, but still enhances the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancer includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0408] Agents that enhance cellular level iRNA uptake can also be added to the pharmaceutical and other compositions of the present invention.For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188 to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97 / 30731 to Lollo et al.) are also known to enhance cellular uptake of dsRNA.Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), iRNAMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent(Roche;Grenzacherstrasse,Switzerland), DOTAP Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), DOSPER Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), or Fugene(Grenzacherstrasse,Switzerland), Transfectam(R) Reagent(Promega;Madison,WI), TransFast(TM)Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass.a D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM) / LipoGen(TM) (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER(TM) transfection Reagent (Genlantis; San Examples of suitable fusion proteins include RiboFect (Bioline; Taunton, MA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA).

[0409] Other agents can be utilized to enhance the penetration of the administered nucleic acid, including glycols, such as ethylene glycol and propylene glycol; pyrroles, such as 2-pyrrole; azone; and terpenes, such as limonene and menthone.

[0410] v. Carrier Certain compositions of the present invention also incorporate a carrier compound into their formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or an analog thereof, that is inert (i.e., has no biological activity itself) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter substance, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory reservoirs, likely due to competition between the carrier compound and the nucleic acid for their normal receptors. For example, recovery of partial phosphorothioate dsRNA in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0411] vi. excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected with the intended mode of administration in mind to provide the desired bulk, consistency, etc., when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (such as starch, sodium starch glycolate, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

[0412] The composition of the present invention can be prepared using pharmaceutically acceptable organic or inorganic excipients that do not adversely react with nucleic acid and are suitable for oral administration.Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone etc.

[0413] The preparation for topical administration of nucleic acid can include sterile and non-sterile aqueous solution, non-aqueous solution in common solvent such as alcohol, or nucleic acid solution in liquid or solid oil base.The solution can also contain buffer, diluent and other suitable additives.Pharmaceutically acceptable organic or inorganic excipients suitable for oral administration that do not adversely react with nucleic acid can be used.

[0414] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0415] vii. Other ingredients The compositions of the present invention may further contain other auxiliary ingredients conventionally found in pharmaceutical compositions at their technically established usage levels.Thus, for example, the compositions may contain additional compatible pharmacologically active ingredients, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, when added, such materials should not excessively interfere with the biological activity of the components of the compositions of the present invention.The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0416] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0417] In some embodiments, pharmaceutical compositions featured herein include (a) one or more iRNA compounds and (b) one or more agents that function via a non-iRNA mechanism and are useful in treating hemolytic disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-lipidemic agents, anti-viral agents, and / or anti-fibrotic agents.

[0418] In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in combination with iRNA as described herein.Other drugs useful for treating liver disease include telbivudine, entecavir, telaprevir and protease inhibitors, such as those disclosed in U.S. Patent Application Publication No. 2005 / 0148548, U.S. Patent Application Publication No. 2004 / 0167116 and U.S. Patent Application Publication No. 2003 / 0144217 by Tung et al.; and U.S. Patent Application Publication No. 2004 / 0127488 by Hale et al.

[0419] The toxicity and therapeutic effect of such compounds can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds that exhibit high therapeutic indices are preferred.

[0420] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of the compositions featured herein generally lies within a range of circulating concentrations, including the ED50, with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods featured herein, a therapeutically effective dose can be estimated initially from cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range (e.g., achieve a reduction in polypeptide concentrations) of the compound, or, if appropriate, of the polypeptide product of the target sequence, including the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms), as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0421] In addition to the administrations discussed above, the iRNAs featured herein may be administered in combination with other known agents effective in treating pathological processes mediated by TTR expression. In any event, the treating physician may adjust the amount and timing of iRNA administration based on the results observed using standard measures of efficacy known in the art or described herein.

[0422] VII. Methods for Inhibiting Expression of TTR The present invention also provides a method for inhibiting expression of transthyretin (TTR) in a cell, comprising contacting the cell with an RNAi agent, e.g., a double-stranded RNAi agent, in an amount effective to inhibit expression of TTR in the cell, thereby inhibiting expression of TTR in the cell.

[0423] Contacting cells with an RNAi agent, for example, a double-stranded RNAi agent, can be carried out in vitro or in vivo. Contacting cells with an RNAi agent in vivo includes contacting cells or a group of cells in a subject, for example, a human subject, with an RNAi agent. A combination of in vitro and in vivo methods of contacting cells or a group of cells is also possible. Contacting cells or a group of cells can be direct or indirect, as discussed above. Furthermore, contacting cells or a group of cells can be carried out via a targeting ligand, for example, any ligand described herein or known in the art. In a preferred embodiment, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand, or any other ligand that directs the RNAi agent to the target site, for example, the liver of a subject.

[0424] The term "inhibit," as used herein, is used interchangeably with "reduce," "silence," "down-regulate," "suppress," and other similar terms, and includes any level of inhibition. Preferably, inhibit includes statistically significant inhibition or clinically significant inhibition.

[0425] The phrase "inhibiting expression of TTR" is intended to refer to the inhibition of expression of any TTR gene (e.g., mouse TTR gene, rat TTR gene, monkey TTR gene, human TTR gene, etc.) and variants or mutants of the TTR gene. Thus, the TTR gene may be a wild-type TTR gene, a mutant TTR gene (such as a mutant TTR gene that causes amyloid deposition), or a transgenic TTR gene in the context of a genetically engineered cell, cell population, or organism.

[0426] "Inhibiting TTR gene expression" includes any level of inhibition of the TTR gene, e.g., at least partial suppression of TTR gene expression. TTR gene expression may be assessed based on the level or change in the level of any variable associated with TTR gene expression, e.g., TTR mRNA level, TTR protein level, or the number or extent of amyloid deposits. The level may be assessed in an individual cell or a group of cells, including, for example, a sample from a subject.

[0427] Inhibition may be assessed by a decrease in the absolute or relative level of one or more variables associated with TTR expression compared t...

Claims

1. A double-stranded ribonucleic acid (RNAi) agent or salt thereof that inhibits expression of transthyretin (TTR) in a cell, wherein the RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region; each of the sense strand and the antisense strand is 21-25 nucleotides in length; the sense strand differs from the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10 by no more than four modified nucleotides, and the antisense strand differs from the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' of SEQ ID NO: 7 by no more than four modified nucleotides; 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; and s is a phosphorothioate linkage. A double-stranded RNAi agent or a salt thereof.

2. 2. The double-stranded RNAi agent or salt thereof of claim 1, wherein the antisense strand differs from the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' of SEQ ID NO: 7 by 3 or less modified nucleotides.

3. 2. The double-stranded RNAi agent or salt thereof of claim 1, wherein the antisense strand differs from the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' of SEQ ID NO:7 by no more than two modified nucleotides.

4. 2. The double-stranded RNAi agent of claim 1, or a salt thereof, wherein the antisense strand differs from the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' of SEQ ID NO:7 by no more than 1 modified nucleotide.

5. 2. The double-stranded RNAi agent or salt thereof of claim 1, wherein the sense strand differs from the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10 by 3 or fewer modified nucleotides.

6. 2. The double-stranded RNAi agent or salt thereof of claim 1, wherein the sense strand differs from the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10 by no more than two modified nucleotides.

7. 2. The double-stranded RNAi agent or salt thereof of claim 1, wherein the sense strand differs from the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10 by no more than 1 modified nucleotide.

8. 2. The double-stranded RNAi agent or salt thereof of claim 1, wherein the sense strand is conjugated to a ligand.

9. 9. The double-stranded RNAi agent or salt thereof of claim 8, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

10. The ligand 【Chemistry 1】 or a salt thereof.

11. 9. The double-stranded RNAi agent or salt thereof of claim 8, wherein the ligand is attached to the 3' end of the sense strand.

12. The RNAi agent is shown in the following schematic diagram: 【Chemistry 2】 (wherein X is O or S) 12. The double-stranded RNAi agent or salt thereof of claim 11, wherein the double-stranded RNAi agent or salt thereof is linked to the ligand as shown in

13. 13. The double-stranded RNAi agent of claim 12, or a salt thereof, wherein X is O.

14. 14. An isolated cell comprising the double-stranded RNAi agent or salt thereof of any one of claims 1-13.

15. A pharmaceutical composition comprising the double-stranded RNAi agent or salt thereof of any one of claims 1 to 13.

16. 16. The pharmaceutical composition of claim 15, comprising a non-buffered solution.

17. 17. The pharmaceutical composition of claim 16, wherein the non-buffered solution is saline or water.

18. 16. The pharmaceutical composition of claim 15, comprising a buffer.

19. 20. The pharmaceutical composition of claim 18, wherein the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

20. 14. An in vitro method for inhibiting transthyretin (TTR) expression in a cell, comprising contacting the cell with the double-stranded RNAi agent of any one of claims 1-13, or a salt thereof, thereby inhibiting expression of the TTR gene in the cell.

21. 21. The method of claim 20, wherein the FAP expression is inhibited by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100%.

22. A pharmaceutical composition for treating a subject suffering from a TTR-related disease, comprising the double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 13.

23. 23. The pharmaceutical composition of claim 22, wherein the Neuropathy Impairment Score (NIS) or modified NIS (mNIS+7) is reduced, slowed, or stopped in a subject suffering from a TTR-related disorder.

24. 23. The pharmaceutical composition of claim 22, wherein the 6-minute walk test (6MWT) is increased in a subject suffering from a TTR-related disorder.

25. 23. The pharmaceutical composition of claim 22, wherein the subject is a human.

26. 23. The pharmaceutical composition of claim 22, wherein the subject has a TTR gene mutation associated with the development of a TTR-related disease.

27. 23. The pharmaceutical composition of claim 22, wherein 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.

28. 23. The pharmaceutical composition of claim 22, wherein the subject has TTR-related amyloidosis.

29. 23. The pharmaceutical composition of claim 22, wherein the subject has TTR-related amyloidosis and the pharmaceutical composition treats polyneuropathy and / or amyloidotic cardiomyopathy of TTR-related amyloidosis.

30. 23. The pharmaceutical composition of claim 22, wherein the double-stranded RNAi agent or salt thereof is administered to the subject by an administration means selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof.

31. 23. The pharmaceutical composition of claim 22, wherein the double-stranded RNAi agent or salt thereof is administered to the subject via subcutaneous administration.

32. 32. The pharmaceutical composition of claim 31, wherein the subcutaneous administration is self-administration.

33. 33. The pharmaceutical composition of claim 32, wherein the self-administration is via a pre-filled syringe or an auto-injector syringe.

34. 23. The pharmaceutical composition of claim 22, wherein the double-stranded RNAi agent or salt thereof is administered chronically to the subject.

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