Transthyretin (TTR) iRNA compositions for treating or preventing TTR-related diseases and methods of using the same

Abstract

To provide iRNA agents, e.g., double stranded iRNA agents, that target the transthyretin (TTR) gene, and methods of using such iRNA agents for treating or preventing TTR-associated diseases.SOLUTION: A double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of TTR in a cell comprises a sense strand complementary to an antisense strand, where the antisense strand comprises a region complementary to a specific sequence, where each strand is about 14 to about 30 nucleotides in length, and where substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.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 Jul. 31, 2015, and U.S. Provisional Patent Application No. 62 / 287,518, filed Jan. 27, 2016. The entire contents of each of the foregoing applications are hereby incorporated by reference herein.

[0002] This application relates to U.S. Provisional Patent Application No. 61 / 881,257, filed Sep. 23, 2013, and International Application PCT / US2014 / 056923, filed Sep. 23, 2014 (the entire contents of each of which are hereby incorporated by reference herein). Further, this application relates to U.S. Provisional Patent Application No. 61 / 561,710, filed Nov. 18, 2011, International Application PCT / US2012 / 065601, filed Nov. 16, 2012, U.S. Provisional Patent Application No. 61 / 615,618, filed Mar. 26, 2012, U.S. Provisional Patent Application No. 61 / 680,098, filed Aug. 6, 2012, U.S. Patent Application No. 14 / 358,972, filed May 16, 2014, and International Application PCT / US2012 / 065691, filed Nov. 16, 2012 (the entire contents of each of which are hereby incorporated by reference herein).

[0003] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format, the entire contents of which are hereby incorporated by reference. The ASCII copy created on Jul. 8, 2016, is named 121301-03020_SL.txt and is 68,289 bytes in size.

Background Art

[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 for retinol (vitamin A) through its association with RBP in blood and CSF. Transthyretin is named for its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apo A-I (the major HDL apolipoprotein), amyloid β peptide, and neuropeptide Y. See (Non-Patent Document 1).

[0005] TTR is a tetramer of four identical 127-amino acid subunits (monomers) that are richly present within a β-sheet structure. Each monomer has two four-stranded β-sheets and an ellipsoidal shape. Antiparallel β-sheet interactions link monomers into dimers. Short loops from each monomer form the major dimer-dimer interactions. These two loop pairs separate the opposing convex β-sheets of the dimer and form 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 characteristic of amyloidosis. Amyloid fibrils consist of misfolded protein aggregates that can result from either overproduction of the precursor protein or specific mutations in the precursor protein. The amyloid-forming ability of TTR may be related to its extensive β-sheet structure; X-ray crystallographic studies have shown that certain amyloid-forming mutations destabilize the protein's tetrameric structure. See, for example, (Non-Patent Document 3).

[0008] Amyloidosis is a general term for a group of amyloid diseases characterized by amyloid deposition. Amyloid diseases are classified based on their precursor proteins. For example, their names start with "A" of amyloid, followed by the abbreviation of the precursor protein. For example, ATTR corresponds to amyloidogenic transthyretin (ibid.).

[0009] There are a very large number of TTR-related diseases, and most of them are amyloid diseases. Wild-type TTR is associated with cardiac amyloidosis in the elderly and is called senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA often involves microscopic depositions in many other organs. TTR amyloidosis appears in various forms. When the peripheral nervous system is more prominently affected, the disease is called familial amyloidotic polyneuropathy (FAP). When mainly the heart is involved but not the nervous system, the disease is called familial amyloid cardiomyopathy (FAC). The third major type of TTR amyloidosis is leptomeningeal amyloidosis (also known as leptomeningeal or meningovascular amyloidosis), central nervous system (CNS) amyloidosis, or amyloidosis type VII. Mutations in TTR can also cause amyloid vitreous opacities, carpal tunnel syndrome, and euthyroid hyperthyroxinaemia (a non-amyloid disease thought to be secondary to an increased association of thyroxine with TTR due to mutant TTR molecules with increased affinity for thyroxine). See, for example, (Non-Patent Document 4).

[0010] Abnormal amyloid-forming proteins can be either hereditary or acquired through somatic mutations (Non-Patent Document 2). Transthyretin-related ATTR is the most common form of hereditary systemic amyloidosis (Non-Patent Document 5). TTR mutations promote the process of TTR amyloid formation and are the most important risk factors in the development of ATTR. More than 85 amyloid-forming TTR variants are known to cause systemic familial amyloidosis. TTR mutations usually cause systemic amyloid deposition, particularly involving the peripheral nervous system, although 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). SSA is estimated to affect more than 25% of the population over 80 years old (Non-Patent Document 7).

Prior Art Documents

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0013] Therefore, in the art, there is a need for an effective treatment for TTR - related diseases. [Means for Solving the Problems]

[0014] The present invention provides an RNAi agent targeting the transthyretin (TTR) gene, such as a double-stranded RNAi agent, and a composition. The present invention also provides a method for inhibiting the expression of TTR and a method for treating or preventing a TTR-related disease in a subject using the RNAi agent of the present invention, such as a double-stranded RNAi agent. The present invention is based, at least in part, on the discovery that an RNAi agent 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 contains 8 or fewer 2'-fluoro modifications on the sense strand, 6 or fewer 2'-fluoro modifications on the antisense strand, 2 phosphorothioate linkages at the 5' end of the sense strand, 2 phosphorothioate linkages at the 5' end of the antisense strand, and a ligand, such as a GalNAc3 ligand, is effective for arresting the activity of the TTR gene. These agents unexpectedly exhibit enhanced TTR gene silencing activity. Without intending to be limited by theory, it is believed that the aforementioned modifications and the combination or partial combination of specific target sites in these RNAi agents confer improved efficacy, stability, potency, and durability to the RNAi agents of the present invention.

[0015] Accordingly, in one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of transthyretin (TTR) in a cell, wherein the RNAi agent comprises a sense strand complementary to an antisense strand, 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 on the sense strand and substantially all of the nucleotides on the antisense strand are modified nucleotides, the sense strand comprises 8 or fewer 2'-fluoro modifications; the antisense strand comprises 6 or fewer 2'-fluoro modifications; the sense strand and the antisense strand each independently comprise 2 phosphorothioate linkages at the 5' end; and the sense strand is bound to at least one ligand.

[0016] In one embodiment, the double-stranded RNAi agent has the formula (IIIe): Sense: 5'-Na -YYY-N b -3’ Antisense: 3’-n p ’-N a ’-Y’Y’Y’-N b ’-5’ (IIIe) (wherein n p ’ is a 2-nucleotide overhang, and n p ’ each nucleotide therein 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 containing 0 to 25 nucleotides, either modified or unmodified or a combination thereof, and each sequence contains 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 represented by.

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

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

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

[0020] The double-stranded region may 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 may have 15 to 30 nucleotides or 19 to 30 nucleotides.

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

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

[0024] The sense strand may contain 2'-fluoro modification of 7 or less, 2'-fluoro modification of 6 or less, 2'-fluoro modification of 5 or less, 2'-fluoro modification of 4 or less, 2'-fluoro modification of 3 or less, or 2'-fluoro modification of 2 or less.

[0025] The antisense strand may contain 2'-fluoro modification of 5 or less, 2'-fluoro modification of 4 or less, 2'-fluoro modification of 3 or less, or 2'-fluoro modification of 2 or less.

[0026] In one embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate or 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 mimic is 5'-vinyl phosphate (5'-VP).

[0028] In one embodiment, the ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched linker. In another embodiment, the ligand is

Chemical formula

[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 attached to a ligand as shown in the following schematic diagram.

Chemical formula

[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 bond; and VP is a 5'-phosphate mimic) comprising 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 bond; and VP is a 5'-phosphate mimetic) and comprises a nucleotide sequence selected from the group consisting of. In another embodiment, the sense and antisense strands are 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 bond). In yet another embodiment, the RNAi agent is selected from any one of the groups of 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 present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of transthyretin (TTR) in cells, wherein the RNAi agent comprises a sense strand complementary to the antisense strand, the antisense strand comprises a region completely 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand contains 2'-fluoro modifications of 8 or less; the antisense strand contains 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently contain two phosphorothioate bonds at the 5' end; and the sense strand is bound to at least one ligand, and the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker.

[0034] In one embodiment, the double-stranded RNAi agent has the formula (IIIe): Sense: 5'-N a -YYY-N b-3' Antisense: 3'-n p '-N a '-Y'Y'Y'-N b '-5' (IIIe) (wherein n p ' is a 2-nucleotide overhang, and n p ' each nucleotide within 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 containing 8-10 nucleotides which are either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, and the modification is a 2'-O-methyl or 2'-fluoro modification) represented by.

[0035] The present invention also provides a cell containing the double-stranded RNAi agent of the present invention, a cell containing the vector of the present invention, and a pharmaceutical composition containing the double-stranded RNAi agent of the present invention or the vector of the present invention.

[0036] In one embodiment, the double-stranded RNAi agent is administered in a non-buffered solution, such as physiological saline or water.

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

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

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

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

[0041] In one embodiment, the subject is suffering from a TTR-related disease.

[0042] In one embodiment, the 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 the double-stranded RNAi agent of the present invention, or the vector of the present invention, or the 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)-related disorder by administering to the subject a prophylactically effective amount of the double-stranded RNAi agent of the present invention, or the vector of the present invention, or the 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 includes administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound to at least one ligand.

[0046] In a further aspect, the present invention provides a method of prophylactically treating a subject at risk of developing a transthyretin (TTR)-related disorder. The method includes administering to the subject a prophylactically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound to at least one ligand.

[0047] In one aspect, the present invention provides a method for reducing, slowing, or halting a neuropathy impairment score (NIS) or revised NIS (mNIS+7) in a subject having a transthyretin (TTR)-related disorder. The method includes administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the present invention, or the vector of the present invention, or the 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 a neuropathy impairment score (NIS) or revised NIS (mNIS+7) in a subject having 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 includes a sense strand complementary to an antisense strand, the antisense strand includes a region complementary to SEQ ID NO: 2, each strand is about 14 to about 30 nucleotides in length, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound to at least one ligand.

[0049] In one aspect, the present invention provides a method for increasing the 6-minute walk test (6MWT) in a subject having a transthyretin (TTR)-related disorder. The method includes administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the present invention, or the vector of the present invention, or the 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 of increasing a six-minute walk test (6MWT) in a subject having a transthyretin (TTR)-related disorder. The method comprises administering to the subject a double-stranded RNAi agent in a therapeutically effective amount, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprises a region complementary to SEQ ID NO: 2, each strand is from about 14 to about 30 nucleotides in length, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand comprises 2'-fluoro modifications of 8 or less; the antisense strand comprises 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at the 5' end; and the sense strand is bound to at least one ligand.

[0051] In one embodiment, the double-stranded RNAi agent has the formula (IIIe): Sense: 5'-N a -YYY-N b -3' Antisense: 3'-n p '-N a '-Y'Y'Y'-N b '-5' (IIIe) (wherein n p ' is a 2 nucleotide overhang, and each nucleotide within n p ' is linked to an adjacent nucleotide via a phosphorothioate linkage; each N a , N b , N b and N b ' independently represents an oligonucleotide sequence comprising from 0 to 25 nucleotides, 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.

[0052] In one embodiment, the subject is 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 having signs or symptoms suggestive of the onset of TTR amyloidosis has a TTR gene mutation associated with the onset of the 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 amyloidotic 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 deposition in the subject.

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

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

[0058] In one embodiment, administration of the double-stranded RNAi agent does not result in an inflammatory response in a subject that is evaluated 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 present invention provides a method of treating a subject having a transthyretin (TTR)-related disorder. The method includes administering to the subject a double-stranded RNAi agent at a fixed 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), wherein the double-stranded RNAi agent includes a sense strand complementary to the antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound 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)-related disorder. The method includes administering to the subject a double-stranded RNAi agent at a fixed 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), wherein the double-stranded RNAi agent includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound 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. 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 includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound 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)-related disorder. The method includes administering to the subject a double-stranded RNAi agent at a dosage 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), where the double-stranded RNAi agent includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound to at least one ligand.

[0063] In another aspect, the present invention provides a method of reducing, slowing, or halting a neuropathy impairment score (NIS) or revised NIS (mNIS+7) in a subject having a transthyretin (TTR)-related disorder. The method includes administering to the subject a double-stranded RNAi agent at a dosage 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 includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2’-fluoro modifications of 8 or less; the antisense strand includes 2’-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5’ end; and the sense strand is bound to at least one ligand.

[0064] In yet another aspect, the present invention provides a method of increasing a six-minute walk test (6MWT) in a subject having a transthyretin (TTR)-related disorder. The method includes administering to the subject a double-stranded RNAi agent at a dosage 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 includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2’-fluoro modifications of 8 or less; the antisense strand includes 2’-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5’ end; and the sense strand is bound to at least one ligand.

[0065] In one aspect, the present invention provides a method for treating a subject having a transthyretin (TTR)-related disorder. The method includes administering to the subject a fixed dose of a double-stranded RNAi agent at 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 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, about 475, about 480, about 490, about 500, about 510, about 520, about 525, about 530, about 540, about 550, about 560, about 570, about 575, about 580, about 590, or about 600 mg), wherein the double-stranded RNAi agent includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes a 2'-fluoro modification of 8 or less; the antisense strand includes a 2'-fluoro modification of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound 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 to the subject a double-stranded RNAi agent in a fixed 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 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, about 475, about 480, about 490, about 500, about 510, about 520, about 525, about 530, about 540, about 550, about 560, about 570, about 575, about 580, about 590, or about 600 mg), wherein the double-stranded RNAi agent includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes 2'-fluoro modifications of 8 or less; the antisense strand includes 2'-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5' end; and the sense strand is bound to at least one ligand.

[0067] In another aspect, the present invention provides a method of reducing, slowing, or halting a neuropathy impairment score (NIS) or revised NIS (mNIS+7) in a subject having a transthyretin (TTR)-related disorder. The method includes administering to the subject a fixed dose of a double-stranded RNAi agent at 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 475, about 480, about 490, about 500, about 510, about 520, about 525, about 530, about 540, about 550, about 560, about 570, about 575, about 580, about 590, or about 600 mg), wherein the double-stranded RNAi agent includes a sense strand complementary to an antisense strand, the antisense strand includes 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 of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand includes a 2’-fluoro modification of 8 or less; the antisense strand includes a 2’-fluoro modification of 6 or less; the sense strand and the antisense strand each independently include two phosphorothioate linkages at the 5’ end; and the sense strand is bound to at least one ligand.

[0068] In yet another aspect, the present invention provides a method of increasing a six-minute walk test (6MWT) in a subject having a transthyretin (TTR)-related disorder. The method comprises administering to the subject a double-stranded RNAi agent in a fixed dose of from about 10 mg to about 600 mg, from about 25 mg to about 500 mg, from about 50 mg to about 500 mg, or from about 80 mg to about 500 mg, from about 25 mg to about 300 mg, from about 50 mg to about 300 mg, or from 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 475, about 480, about 490, about 500, about 510, about 520, about 525, about 530, about 540, about 550, about 560, about 570, about 575, about 580, about 590, or about 600 mg), wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprises a region complementary to SEQ ID NO: 2 (5’-UGGGAUUUCAUGUAACCAAGA-3’), each strand is from about 14 to about 30 nucleotides in length, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand comprises 2’-fluoro modifications of 8 or less; the antisense strand comprises 2’-fluoro modifications of 6 or less; the sense strand and the antisense strand each independently comprise two phosphorothioate linkages at the 5’ end; and the sense strand is bound to at least one ligand.

[0069] In one embodiment, the double-stranded RNAi agent has the formula (IIIe): Sense: 5’-N a -YYY-N b -3’ Antisense: 3’-n p ’-Na ’-Y’Y’Y’-N b ’-5’ (IIIe) (wherein, n p ’ is a 2-nucleotide overhang, and n p each nucleotide within ’ is linked to an adjacent nucleotide via a phosphorothioate bond; each N a 、N b 、N b and N b ’ independently represents an oligonucleotide sequence containing 0 to 25 nucleotides which are either modified or unmodified or a combination thereof, and each sequence contains 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 represented 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 bond; and VP is a 5’-phosphate mimic) and contains 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 bond; and VP is a 5’ phosphate mimic) and comprises a nucleotide sequence selected from the group consisting of.

[0072] In one embodiment, the sense and antisense strands are 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 bond).

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

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

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

[0076] In one embodiment, the double-stranded RNAi agent is chronically administered 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 having a family history of a TTR-related disease, or a subject having signs or symptoms suggesting the onset of TTR amyloidosis has a TTR gene mutation associated with the onset 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 amyloidotic polyneuropathy (FAP), familial amyloid 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 deposition in the subject.

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

[0082] In one embodiment, the method further comprises the step of evaluating 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 that is evaluated 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, the agent suitable for use in the method of the present invention is AD-65492. AD-65492 may be chronically administered to the subject every 4 weeks, every 5 weeks, or every 6 weeks, or quarterly.

[0085] In one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent used when inhibiting the expression of transthyretin (TTR) intracellularly. The agent contains a sense strand complementary to the antisense strand, where the sense and antisense strands contain a nucleotide sequence selected from the group consisting of any of the nucleotide sequences in Table 5.

[0086] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for use in inhibiting the expression of transthyretin (TTR) in cells. The agent includes a sense strand complementary to the antisense strand, where the antisense strand includes a complementary region of at least 15 contiguous nucleotides that differs from any one of the antisense sequences in Table 6 by 3 or fewer nucleotides, where substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; and the sense strand is bound to at least one ligand.

[0087] The sense and antisense strands may include 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 present invention provides a double-stranded ribonucleic acid (RNAi) agent for use in inhibiting the expression of transthyretin (TTR) in cells, where the RNAi agent includes a sense strand complementary to the antisense strand, the sense strand includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand includes the nucleotide sequence 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 bond).

[0089] In another aspect, the present invention provides a method for treating a subject suffering from a TTR-related disease. The method includes administering a double-stranded RNAi agent to the subject at a dose of about 50 mg to about 300 mg, wherein the RNAi agent includes a sense strand complementary to an antisense strand, the sense strand includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand includes the nucleotide sequence 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 bond), thereby treating a 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 disease. The method includes administering a double-stranded RNAi agent to the subject at a dose of about 50 mg to about 300 mg, wherein the RNAi agent includes a sense strand complementary to an antisense strand, the sense strand includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand includes the nucleotide sequence 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 bond), thereby prophylactically treating a subject at risk of developing a TTR-related disease.

[0091] In one aspect, the present invention provides a method for reducing, slowing, or halting a neuropathy impairment score (NIS) or revised NIS (mNIS+7) in a subject suffering from or at risk of developing a TTR-related disease. The method includes administering to the subject a double-stranded RNAi agent at a dose of about 50 mg to about 300 mg, wherein the RNAi agent includes a sense strand complementary to an antisense strand, the sense strand includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand includes the nucleotide sequence 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 bond), 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 for increasing a six-minute walk test (6MWT) in a subject suffering from or at risk of developing a TTR-related disease. The method includes administering to the subject a double-stranded RNAi agent at a dose of about 50 mg to about 300 mg, wherein the RNAi agent includes a sense strand complementary to an antisense strand, the sense strand includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand includes the nucleotide sequence 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 bond), thereby increasing the six-minute walk test (6MWT) in a subject suffering from or at risk of developing a TTR-related disease.

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

Brief Description of the Drawings

[0094]

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[0095] ​The present invention provides an RNAi agent that targets the transthyretin (TTR) gene, such as a double-stranded RNAi agent, and a composition. The present invention also provides a method for inhibiting the expression of TTR, and a method for treating or preventing a TTR-related disease in a subject using the RNAi agent of the present invention, such as a double-stranded RNAi agent. The present invention is based, at least in part, on the discovery that an RNAi agent 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 has 8 or fewer 2'-fluoro modifications (e.g., 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications) on the sense strand, 6 or fewer 2'-fluoro modifications (e.g., 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications) on the antisense strand, 2 phosphorothioate bonds at the 5' end of the sense strand, 2 phosphorothioate bonds at the 5' end of the antisense strand, and a ligand, such as a GalNAc3 ligand, is effective for selectively arresting the activity of the TTR gene. These agents surprisingly exhibit enhanced TTR gene silencing activity. Without intending to be limited by theory, it is believed that the aforementioned modifications and combinations or partial combinations of specific target sites in these RNAi agents confer improved efficacy, stability, potency, and durability to the RNAi agents of the present invention.

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

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

[0098] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the articles for grammatical purposes. As an example, "an element" means one element, or two or more elements such as, for example, a plurality of elements.

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

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

[0101] The term "about" is used herein to mean within the typical ranges acceptable in the art. For example, "about" can be understood to be within about two 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 the range.

[0102] As used herein, "transthyretin" ("TTR") refers to a well-known gene and protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter for 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 aggregate to form amyloid fibrils that are extracellular deposits and can cause amyloidosis. See, for example, Saraiva M.J.M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11 for a review. Molecular cloning and the 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 the human TTR mRNA transcript 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 accessible 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 long enough to serve as a substrate for iRNA-directed cleavage in or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the TTR gene. In one embodiment, the target sequence is present within the protein-coding region of the TTR gene. In another embodiment, the target sequence is present within the 3' UTR of the TTR gene.

[0104] The target sequence may be about 9 to 36 nucleotides in length, such as about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides in length, such as 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 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 those recited above are also considered to be part of the present invention.

[0105] It should be noted that there is a misspelling in the original text, "embodment" should be "embodiment".In some embodiments of the present invention, the target sequence of the TTR gene comprises nucleotides 615-637 of SEQ ID NO: 1 or nucleotides 505-527 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, using standard nucleotide nomenclature, includes the nucleotide strand described by the recited sequence.

[0107] "G", "C", "A", "T", and "U" typically represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, the terms "ribonucleotide" or "nucleotide" are also understood to refer to modified nucleotides or alternative substituents as further detailed below (see, for example, Table 2). One of ordinary skill in the art is well aware that guanine, cytosine, adenine, and uracil can be replaced with other moieties in an oligonucleotide containing nucleotides with such substituents without substantially altering the base pairing properties of the oligonucleotide. By way of non-limiting example, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced with a nucleotide containing inosine, for example, in the nucleotide sequence of the dsRNA taken up in the present invention. In another example, adenine and cytosine can be replaced with guanine and uracil, respectively, anywhere in the oligonucleotide to form G-U wobble base pairs with the target mRNA. Sequences containing such substituents are suitable for the compositions and methods taken up in the present invention.

[0108] The terms "iRNA", "RNAi agent", "iRNA agent", and "RNA interference agent" are used synonymously herein and refer to an agent that contains RNA as defined herein and mediates the 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, for example, inhibits TTR gene expression in cells such as cells within a subject such as a mammalian subject.

[0109] In one embodiment, examples of the RNAi agent of the present invention include single-stranded RNA that interacts with a target RNA sequence such as a TTR target mRNA sequence and induces cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded by type III endonucleases known as Dicer, yielding short double-stranded interfering RNA (siRNA) containing sense and antisense strands (Sharp et al. (2001) Genes Dev. 15:485). The ribonuclease III-like enzyme of Dicer processes these dsRNAs to yield short interfering RNAs of 19-23 base pairs characterized by 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex and allow the complementary antisense strand to guide 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 to induce 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 generated inside cells and promotes the formation of the RISC complex to silence the target gene, i.e., the TTR gene. Thus, 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 the target mRNA. The single-stranded RNAi agent binds to Argonaute 2 of the RISC endonuclease and then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides and are 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, which are incorporated herein by reference in their entireties. Any antisense nucleotide sequence described herein may be used as a single-stranded siRNA described herein or chemically modified by the method 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, referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a ribonucleic acid molecular complex having a double-stranded structure that includes two anti-parallel and substantially complementary nucleic acid strands that 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, the double-stranded RNA (dsRNA) causes degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0112] Generally, most of the nucleotides of each strand of the dsRNA molecule are ribonucleotides, but as detailed herein, each strand or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Further, as used throughout this application, an "RNAi agent" may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications to multiple nucleotides.

[0113] As used herein, the term "modified nucleotide" independently refers to a nucleotide having a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removals of, for example, functional groups or atoms to the internucleoside linkage, the sugar moiety, or the nucleobase. Modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modification is encompassed by the "RNAi agent" when used in an siRNA-type molecule for the purposes of the present application and claims.

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

[0115] The two strands forming the double-stranded structure may be different parts of a larger RNA molecule or they may be separate RNA molecules. When the two strands are parts of one larger molecule and are thus joined by a nucleotide strand that is uninterrupted between the 3’ end of one strand forming the double-stranded structure and the 5’ end of each other strand, the joined RNA strands are referred to as a “hairpin loop”. The hairpin loop may contain at least one unpaired nucleotide; in some embodiments, the 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, at least 23 or more unpaired nucleotides. In some embodiments, the hairpin loop may be 10 or fewer nucleotides. In some embodiments, the hairpin loop may be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop may be 4 to 10 unpaired nucleotides. In some embodiments, the hairpin loop may be 4 to 8 nucleotides.

[0116] When the two substantially complementary strands of dsRNA are constituted by separate RNA molecules, these molecules may be covalently linked, but are not necessarily so. When the two strands are covalently joined by means other than an uninterrupted nucleotide strand between the 3’ end of one strand forming the double-stranded structure and the 5’ end of each other strand, the joined structure is referred to as a “linker”. The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the double-stranded structure, RNAi may include one or more nucleotide overhangs.

[0117] In one embodiment, the RNAi agent of the present invention is a dsRNA that interacts with a target RNA sequence, such as a TTR target mRNA sequence, to induce cleavage of the target RNA, and each strand thereof is 24 to 30 nucleotides in length. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by type III endonucleases known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The ribonuclease III-like enzyme of Dicer processes dsRNA to yield short interfering RNAs of 19 to 23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex to enable 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 to induce 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, such as 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, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise overhangs of at least one nucleotide.

[0118] In one embodiment, the RNAi agent of the present invention is a dsRNA that interacts with the TTR RNA sequence to induce cleavage of the target RNA, and each strand thereof contains 19 to 23 nucleotides. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by type III endonucleases known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The ribonuclease III-like enzyme of Dicer processes the dsRNA to yield short interfering RNAs of 19 to 23 base pairs with characteristic 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling 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 to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the present invention is a dsRNA of 24 to 30 nucleotides 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, for example, dsRNA. For example, when the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa, there is a nucleotide overhang. The dsRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleotides. The nucleotide overhang may include, or consist of, nucleotide / nucleoside analogs including, but not limited to, deoxyribonucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotides of the overhang may be present at the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA. In one embodiment of the dsRNA, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, such as 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 contains a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand contains a 5' overhang of at least two nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' and 5' ends of one strand of the RNAi agent contain an overhang of at least one nucleotide.

[0120] In one embodiment, the antisense strand of the dsRNA has overhangs, for example, at the 3' end and / or the 5' end, for example, 1 to 10 nucleotides such as 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has overhangs, for example, at the 3' end and / or the 5' end, 1 to 10 nucleotides such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with phosphorothioate nucleosides.

[0121] In certain embodiments, the overhangs in the sense strand or the antisense strand, or both, can include a nucleotide length extended longer than 10, for example, a 1 to 30 nucleotide length, a 2 to 30 nucleotide length, a 10 to 30 nucleotide length, or a 10 to 15 nucleotide length. In certain embodiments, the extended overhang is present on the double-stranded sense strand. In certain embodiments, the extended overhang is present at the 3' end of the double-stranded sense strand. In certain embodiments, the extended overhang is present at the 5' end of the double-stranded sense strand. In certain embodiments, the extended overhang is present on the double-stranded antisense strand. In certain embodiments, the extended overhang is present at the 3' end of the double-stranded antisense strand. In certain embodiments, the extended overhang is present at the 5' end of the double-stranded antisense strand. In certain embodiments, one or more of the nucleotides in the overhang are replaced with nucleoside phosphorothioates. In certain embodiments, the overhang includes a self-complementary portion such that the overhang has the ability to form a stable hairpin structure under physiological conditions.

[0122] "Smooth" or "blunt-ended" means that there are no unpaired nucleotides at the ends of the double-stranded RNAi agent, i.e., there are no nucleotide overhangs. A "blunt-ended" RNAi agent is double-stranded over its entire length, i.e., a dsRNA that has no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents having a nucleotide overhang at one end (i.e., an agent having one overhang and one blunt end) or RNAi agents having nucleotide overhangs at both ends.

[0123] The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., a dsRNA, that contains a region substantially complementary to a target sequence, e.g., the TTR mRNA. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., the TTR nucleotide sequence, as defined herein. If the complementarity of the complementary region to the target sequence is not perfect, the mismatches may be present within the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are present within the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotide at the 5' end and / or 3' end of the iRNA. In one embodiment, the double-stranded RNAi agent of the present invention contains nucleotide mismatches within the antisense strand. In another embodiment, the double-stranded RNAi agent of the present invention contains nucleotide mismatches within the sense strand. In one embodiment, the nucleotide mismatches are present, for example, within the 5, 4, 3, 2, or 1st nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are present, for example, within the 3' terminal nucleotide of the iRNA.

[0124] The term "sense strand" or "passenger strand" refers to a strand of an iRNA that contains a region substantially complementary to the region of the antisense strand as defined herein when used herein.

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

[0126] As used herein, unless otherwise specified, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, as would be understood by one of ordinary skill in the art, refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under specific conditions to produce a double-stranded structure. Such conditions can be, for example, stringent conditions, which include 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, 12 - 16 hours at 50 °C or 70 °C, followed by washing (see, for example, "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 also be applied. One of ordinary skill in the art can determine the optimal set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotides.

[0127] For example, the complementary sequences within iRNAs, such as within the dsRNAs described herein, include base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence, spanning all or part 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 5, 4, 3, or 2 or fewer, mismatched base pairs while maintaining the ability to hybridize under the conditions most appropriate for their ultimate use, such as inhibition of gene expression through the RISC pathway, during hybridization of up to 30 base pairs of duplex. However, when two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches with respect to the determination of complementarity. For example, a dsRNA comprising one 21-nucleotide-long oligonucleotide and another 23-nucleotide-long oligonucleotide, wherein the longer oligonucleotide comprises a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, is still referred to as "fully complementary" for the purposes described herein.

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

[0129] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used with respect to base pairing between the sense and antisense strands of a dsRNA or between the antisense strand of an iRNA agent and a target sequence, as would 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 the target mRNA (e.g., the mRNA encoding the TTR gene). For example, a polynucleotide is complementary to at least a portion of the TTR mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the TTR gene.

[0131] Accordingly, 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 the target TTR sequence and are at least about 80% complementary over their entire length to a corresponding region of any one nucleotide sequence of SEQ ID NO: 2 (5'-UGGGAUUUCAUGUAACCAAGA-3') or to a fragment of any one of SEQ ID NOs: 1, 2, and 5, 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, and include adjacent nucleotide sequences.

[0133] In one embodiment, the RNAi agent of the present invention includes a sense strand that is substantially complementary to an antisense polynucleotide (and further the antisense polynucleotide is complementary to a target TTR sequence), wherein the sense strand polynucleotide is at least about 80% complementary over its entire length to a corresponding region of any one of the nucleotide sequences in Tables 1, 3, 5, 6, and 7, for example, 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 contiguous nucleotide sequences.

[0134] In another embodiment, the RNAi agent of the present invention includes an antisense strand that is substantially complementary to a target TTR sequence, and is at least about 80% complementary over its entire length to a corresponding region of any one of the nucleotide sequences in Tables 1 and 3, for example, 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 contiguous nucleotide sequences.

[0135] In some embodiments, generally, most of the nucleotides of each strand are ribonucleotides, but as described in detail herein, one or both strands may also include one or more non-ribonucleotides such as, for example, deoxyribonucleotides and / or modified nucleotides. Further, "iRNA" includes ribonucleotides with chemical modifications. Such modifications include all types of modifications disclosed herein or known in the art. For the purposes of this specification and the claims, any such modification is included within "iRNA" in the context of its use in an iRNA molecule.

[0136] In one aspect of the present invention, the agent used in the method and composition 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 the sequence within the target mRNA. The single-stranded antisense oligonucleotide can base pair with the mRNA and inhibit translation in a stoichiometric manner by physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule is about 15 to about 30 nucleotides in length and may 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] "TTR-related disease", as used herein, is intended to include any disease associated with the TTR gene or protein. Such diseases may be caused, for example, by overproduction of the TTR protein, by TTR gene mutations, by abnormal cleavage of the 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 deposits. TTR-related diseases include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, amyloid vitreous opacities, carpal tunnel syndrome, and hyperthyroxinemia. The symptoms of TTR amyloidosis include sensory neuropathy (e.g., sensory disturbances, sensory hypoesthesia in the distal extremities), autonomic neuropathy (e.g., gastrointestinal disturbances, such as gastric ulcers, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacities, renal failure, nephrosis, substantially decreased mBMI (modified body mass index), cranial neuropathy, and lattice corneal dystrophy.

[0138] II. The iRNA of the present 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 intracellularly, such as in cells within a subject, such as a mammal, such as a human having a TTR-related disease. The dsRNA comprises an antisense strand having a complementary region that is at least partially complementary to at least a portion of the mRNA formed during TTR gene expression. The complementary region has a length of about 30 nucleotides or less (e.g., a length of about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less). Upon contact with cells that express the TTR gene, the iRNA selectively inhibits the expression of the TTR gene (e.g., human, primate, non-primate, or avian Sertpinc1 gene) by at least about 10% in an assay by, for example, a PCR or branched DNA (bDNA)-based method, or a protein-based method such as immunofluorescence analysis using, for example, Western blot or flow cytometry techniques.

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

[0140] Generally, the double-stranded structure has a length of 15 to 30 base pairs, such as 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the present invention.

[0141] Similarly, the complementary region of the target sequence has a length of 15 to 30 nucleotides, such as 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the present 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 dsRNA longer than about 21-23 nucleotides in length may serve as a substrate for Dicer. As will be appreciated by those skilled in the art, the target region of the RNA to be cleaved is, in most cases, part of a larger RNA molecule, which is often an mRNA molecule. Where applicable, the "portion" of the mRNA target is a continuous sequence of the mRNA target that is long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage through the RISC pathway).

[0143] Those skilled in the art will also recognize that double-stranded regions such as double-stranded regions of about 9 to 36 base pairs, such as 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 33, 15 to 33, 9 to 32, 10 to 32, 11 to 32, 12 to 32, 13 to 32, 14 to 32, 15 to 32, 9 to 31, 10 to 31, 11 to 31, 12 to 31, 13 to 32, 14 to 31, 15 to 31, 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 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs are the main functional part of the dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region exceeding 30 base pairs is dsRNA, to the extent that it is processed, for example, into a 15 to 30 base pair functional double strand targeting a desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA. In another embodiment, the dsRNA is not a natural miRNA. In another embodiment, the iRNA agent useful for targeting TTR expression is not generated in target cells by cleavage of larger dsRNAs.

[0144] The dsRNA described in this specification may further include one or more single-stranded nucleotide overhangs, such as, for example, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may unexpectedly have superior inhibitory properties compared to their blunt-end counterparts. Nucleotide overhangs may include, or consist of, nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotides of the overhang may be present on the 5′ end, the 3′ end, or both ends of either the antisense or sense strand of the dsRNA. In certain embodiments, longer, extended overhangs are possible.

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

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

[0147] In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences of a sense sequence and an antisense sequence. The sense strand is selected from the group of sequences provided by any one of Tables 1, 3, 5, 6, and 7, and the antisense strand corresponding to the sense strand is selected from the group of sequences of any one of Tables 1, 3, 5, 6, and 7. In this aspect, 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 that occurs during TTR gene expression. Thus, in this aspect, the dsRNA comprises two oligonucleotides, one oligonucleotide is described as any one of the sense strands of Tables 1, 3, 5, 6, and 7, and the second oligonucleotide is described as the antisense strand corresponding to any one of the sense strands 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] Some of the sequences of Tables 1, 3, 5, 6, and 7 are described as modified and / or conjugated sequences, but it is understood that the RNA of the iRNA of the present invention, such as the dsRNA of the present invention, may include any one of the sequences described in Tables 1, 3, 5, 6, and 7 that is unmodified, non-conjugated, and / or modified and / or conjugated differently from that described.

[0149] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of about 20-23 base pairs, such as 21 base pairs, are particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, other workers in the art 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 include at least one strand that is at least 21 nucleotides in length. It can be reasonably expected that shorter double-strands having one of the sequences of any one of Tables 1, 3, 5, 6, and 7, with only a few nucleotides missing at one or both ends, may be similarly effective compared to the dsRNAs described above. Accordingly, having a sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides derived from one of the sequences of any one of Tables 1, 3, 5, 6, and 7 and having the ability to inhibit TTR gene expression, dsRNAs that differ from the dsRNA containing the full-length sequence by about 5, 10, 15, 20, 25, or 30% or less 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 TTR transcripts that are highly sensitive to RISC-mediated cleavage. Accordingly, the present invention further features iRNAs that target within one of these sequences. In the usage herein, when an iRNA promotes cleavage of a transcript anywhere within a particular site, the iRNA is said to target within that particular site of the RNA transcript. Such iRNAs generally contain about 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 sequences in the TTR gene.

[0151] Target arrays are generally about 15 to 30 nucleotides in length, but there is wide diversity in the suitability of specific sequences within this range to induce cleavage of any given target RNA. The various software packages and guidelines presented herein provide guidance for identifying optimal target arrays for any given gene target, but an empirical approach can also be taken of actually or metaphorically (including, for example, by computer simulation) placing a "window" or "mask" of a given size (21 nucleotides as a non-limiting example) over the target RNA sequence to identify sequences within a size range that can serve the role of the target sequence. By continuously moving the sequence "window" one nucleotide upstream or downstream of the first target sequence position, potential next target sequences can be identified until a complete set of all possible sequences for any given target size that has been selected is identified. This process, combined with the systematic synthesis of the identified sequences and testing to identify sequences that function optimally (using assays described herein or known in the art), can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, for example, sequences identified in any one of Tables 1, 3, 5, 6, and 7 represent effective target sequences, while further optimization of inhibition efficiency can be considered by identifying sequences with equivalent or better inhibition characteristics by "walking the window" continuously one nucleotide upstream or downstream of a given sequence.

[0152] Furthermore, nucleotides are systematically added or removed to create longer or shorter sequences, and from that position, the created sequences are tested by walking a window of a size longer or shorter than the target RNA, whereby further optimization of any sequence, for example, identified in any one of Tables 1, 3, 5, 6, and 7, can be achieved. Also in this case, by combining the approach for creating this new target candidate with the test of the effectiveness of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein, a further improvement in the inhibition efficiency can be brought about. Still further, for example, by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or contemplated herein, molecules can be further optimized as expression inhibitors (e.g., increased serum stability or circulation half-life, increased thermal stability, promotion of transmembrane delivery, targeting of specific positions or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes, etc.), whereby such optimized sequences can be regulated.

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

[0154] III. Modified iRNAs of the Invention In one embodiment, the RNA of the iRNA of the present invention, such as dsRNA for example, is unmodified and does not contain chemical modifications and / or linkages that are known in the art and described herein. In another embodiment, the RNA of the iRNA of the invention, such as dsRNA for example, is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all of the nucleotides of the iRNA of the present invention are modified. In some embodiments, substantially all of the nucleotides of the iRNA of the present invention are modified and the iRNA contains 2'-fluoro modifications of 8 or less (e.g., 2'-fluoro modifications of 7 or less, 2'-fluoro modifications of 6 or less, 2'-fluoro modifications of 5 or less, 2'-fluoro modifications of 4 or less, 2'-fluoro modifications of 3 or less, or 2'-fluoro modifications of 2 or less) on the sense strand and 2'-fluoro modifications of 6 or less (e.g., 2'-fluoro modifications of 5 or less, 2'-fluoro modifications of 4 or less, 2'-fluoro modifications of 3 or less, or 2'-fluoro modifications of 2 or less) on the antisense strand. In other embodiments, all of the nucleotides of the iRNA of the present invention are modified and the iRNA contains 2'-fluoro modifications of 8 or less (e.g., 2'-fluoro modifications of 7 or less, 2'-fluoro modifications of 6 or less, 2'-fluoro modifications of 5 or less, 2'-fluoro modifications of 4 or less, 2'-fluoro modifications of 3 or less, or 2'-fluoro modifications of 2 or less) on the sense strand and 2'-fluoro modifications of 6 or less (e.g., 2'-fluoro modifications of 5 or less, 2'-fluoro modifications of 4 or less, 2'-fluoro modifications of 3 or less, or 2'-fluoro modifications of 2 or less) on the antisense strand. The iRNA of the present invention where "substantially all of the nucleotides are modified" is modified on a large scale, but not completely, and may contain 5 or less, 4, 3, 2, or 1 unmodified nucleotides.

[0155] The nucleic acids addressed by 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.L. 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, conjugation, inversion), or 3′ end modifications (conjugation, DNA nucleotides, inversion, etc.); base modifications such as substitution with a stabilizing base, a destabilizing base, or a base that base pairs with an expanded partner repertoire, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′ or 4′ position) or sugar substitutions; backbone modifications including modification or substitution of the phosphodiester bond. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing a modified backbone or RNAs lacking native internucleoside linkages. RNAs having a modified backbone include, in particular, those having no phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs having no phosphorus atom in their internucleoside backbone are also considered oligonucleosides. In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0156] Examples of modified RNA backbones include phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, 3'-alkylene phosphonate and chiral phosphonate including methyl and other alkyl phosphonates, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and normal 3'-5' linkages, boranophosphate having 2'-5' linked analogs thereof, and boranophosphate having reverse polarity where the adjacent nucleoside unit pairs are linked 3'-5' to 5'-3', or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0157] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent No. 3,687,808; U.S. Patent No. 4,469,863; U.S. Patent No. 4,476,301; U.S. Patent No. 5,023,243; U.S. Patent No. 5,177,195; U.S. Patent No. 5,188,897; U.S. Patent No. 5,264,423; U.S. Patent No. 5,276,019; U.S. Patent No. 5,278,302; U.S. Patent No. 5,286,717; 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. 5,571,799; U.S. Patent No. 5,587,361; U.S. Patent No. 5,625,050; U.S. Patent No. 6,028,188; U.S. Patent No. 6,124,445; U.S. Patent No. 6,160,109; U.S. Patent No. 6,169,170; U.S. Patent No. 6,172,209; U.S. Patent No. 6,239,265; U.S. Patent No. 6,277,603; U.S. Patent No. 6,326,199; U.S. Patent No. 6,346,614; U.S. Patent No. 6,444,423; U.S. Patent No. 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, each of which is hereby incorporated by reference in its entirety.

[0158] The modified RNA backbone that does not contain a phosphorus atom therein has a backbone formed by short-chain alkyl or cycloalkyl nucleoside internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside internucleoside linkages, or one or more short-chain heteroatom or heterocyclic nucleoside internucleoside linkages. These include morpholino linkages (some of which are formed from the sugar moiety 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 having amide backbones; and others having mixed N, O, S, and CH2 components.

[0159] Representative U.S. patents that teach the preparation of the above oligonucleosides include U.S. Patent No. 5,034,506; U.S. Patent No. 5,166,315; 5,185,444; U.S. Patent No. 5,214,134; U.S. Patent No. 5,216,141; U.S. Patent No. 5,235,033; U.S. Patent No. 5,64,562; U.S. Patent No. 5,264,564; U.S. Patent No. 5,405,938; U.S. Patent No. 5,434,257; U.S. Patent No. 5,466,677; U.S. Patent No. 5,470,967; U.S. Patent No. 5,489,677; U.S. Patent No. 5,541,307; U.S. Patent No. 5,561,225; U.S. Patent No. 5,596,086; U.S. Patent No. 5,602,240; U.S. Patent No. 5,608,046; U.S. Patent No. 5,610,289; U.S. Patent No. 5,618,704; U.S. Patent No. 5,623,070; U.S. Patent No. 5,663,312; U.S. Patent No. 5,633,360; U.S. Patent No. 5,677,437; and U.S. Patent No. 5,677,439, each of which is hereby incorporated herein by reference in its entirety, but is not limited thereto.

[0160] In another embodiment, suitable RNA mimics are considered for use in iRNA, in which both the sugar and the internucleoside linkage, i.e., the backbone of the nucleotide unit, are replaced with new groups. The base units are maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly attached to the azanitrogen 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. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, the entire contents of each of which are incorporated herein by reference. Further, PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0161] Some embodiments contemplated by the present invention include oligonucleosides having an RNA with a phosphorothioate backbone, and in particular, --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [known as the methylene(methylimino) or MMI backbone] of U.S. Patent No. 5,489,677 mentioned above, --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [the natural phosphodiester backbone is represented as --O--P--O--CH2--], and an amide backbone of U.S. Patent No. 5,602,240 mentioned above. In some embodiments, the RNA contemplated herein has the morpholino backbone structure of U.S. Patent No. 5,034,506 mentioned above.

[0162] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs such as the dsRNAs discussed herein can contain at the 2'-position one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C 10 alkyl, or C2-C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2 (where n and m are from 1 to about 10). In another embodiment, the dsRNA contains at the 2'-position one of: C1-C 10 lower alkyl, substituted lower alkyl, aralkyl, aralkyl, O-aralkyl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaararyl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group that improves the pharmacokinetic properties of the iRNA, or group that improves the pharmacodynamic properties of the iRNA, and other substituents having similar properties. In some embodiments, the modification is 2'-methoxyethoxy (also known as 2'-O-CH2CH2OCH3, 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., it contains an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE as described in the examples hereinbelow, i.e., the O(CH2)2ON(CH3)2 group, 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.

[0163] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the iRNA, specifically at the 3'-position of the sugar on the 3'-terminal nucleotide, or in the 2'-5' linked dsRNA, and at the 5'-position of the 5'-terminal nucleotide. The iRNA may also have a sugar mimic such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents that teach the preparation of the above modified sugar structures include, among others, U.S. Patent No. 4,981,957; U.S. Patent No. 5,118,800; U.S. Patent No. 5,319,080; U.S. Patent No. 5,359,044; U.S. Patent No. 5,393,878; U.S. Patent No. 5,446,137; U.S. Patent No. 5,466,786; U.S. Patent No. 5,514,785; U.S. Patent No. 5,519,134; U.S. Patent No. 5,567,811; U.S. Patent No. 5,576,427; U.S. Patent No. 5,591,722; U.S. Patent No. 5,597,909; U.S. Patent No. 5,610,300; U.S. Patent No. 5,627,053; U.S. Patent No. 5,639,873; U.S. Patent No. 5,646,265; U.S. Patent No. 5,658,873; U.S. Patent No. 5,670,633; and U.S. Patent No. 5,700,920, which are hereby incorporated herein by reference in their entireties.

[0164] The RNA of the iRNA of the present invention may also include nucleobase (often simply referred to as "base" in the art) modifications or substitutions. In the usage of this specification, "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-azauracil, 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.Furthermore, as nucleobases, those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.L, ed. John Wiley & Sons, 1990, those disclosed 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, S.T. and Lebleu, B., Ed., CRC Press, 1993 may be mentioned. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6 to 1.2 °C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), is a exemplary base substitution, and even more particularly so when combined with 2'-O-methoxyethyl sugar modification.

[0165] Representative U.S. patents that teach the preparation of the above-mentioned specific modified nucleobases as well as other modified nucleobases, which are hereby incorporated herein by reference in their entireties, include U.S. Patent No. 3,687,808, U.S. Patent No. 4,845,205; U.S. Patent No. 5,130,30; U.S. Patent No. 5,134,066; U.S. Patent No. 5,175,273; U.S. Patent No. 5,367,066; U.S. Patent No. 5,432,272; U.S. Patent No. 5,457,187; U.S. Patent No. 5,459,255; U.S. Patent No. 5,484,908; U.S. Patent No. 5,502,177; U.S. Patent No. 5,525,711; U.S. Patent No. 5,552,540; U.S. Patent No. 5,587,469; U.S. Patent No. 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, but are not limited thereto.

[0166] The RNA of the iRNA can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, the agent of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety such that the ribose moiety includes an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide that includes a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose within the 3'-end conformation. The addition of locked nucleic acids 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, O.R. 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 polynucleotides of the present invention include, but are not limited to, nucleosides that include a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides that include a 4' to 2' bridge.Examples of such bicyclic nucleosides bridged from the 4' to the 2' position include, but are not limited to, 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2'(also referred to as "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2'(and its analogs; see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2'(and its analogs; see, e.g., U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2'(and its analogs; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-O-N(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. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2'(see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2'(and its analogs; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated by reference herein.

[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, namely, U.S. Patent No. 6,268,490; No. 6,525,191; No. 6,670,461; No. 6,770,748; No. 6,794,499; No. 6,998,484; No. 7,053,207; No. 7,034,133; No. 7,084,125; No. 7,399,845; No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 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 aforementioned bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

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

[0170] The iRNA of the present invention may also contain one or more "stereostructurally 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. CRNs lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker positions oxygen in an optimal position for stability and affinity and has a length sufficient to result in a reduction in the packing of the ribose ring.

[0171] Representative publications teaching 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 WO 2013 / 036868 (the entire contents of each are hereby incorporated by reference herein).

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

[0173] Representative U.S. publications teaching the preparation of UNA 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 are hereby incorporated by reference herein).

[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'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3”-phosphate, inverted base dT (idT), and the like. The disclosure of this modification is in WO 2011 / 005861 pamphlet.

[0175] Other modifications of the nucleotides of the iRNA of the present invention include a 5' phosphate or 5' phosphate mimetic on the antisense strand of the RNAi agent, such as a 5' terminal phosphate or phosphate mimetic. Suitable phosphate mimetics are disclosed, for example, in US Patent Application Publication No. 2012 / 0157511, the entire content of which is incorporated herein by reference.

[0176] A. Modified iRNA Containing the Motif of the Present Invention In certain embodiments of the present invention, the double-stranded RNAi agent of the present invention comprises chemical modifications as disclosed, for example, in US Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, or International Application PCT / US2012 / 065691, filed November 16, 2012, the entire content of each of which is incorporated herein by reference.

[0177] More specifically, it has been unexpectedly discovered that when the sense strand and the antisense strand of the 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] Accordingly, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the TTR gene) in vivo. The RNAi agent includes 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 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 the antisense strand typically form double-stranded RNA (dsRNA) as a double strand, also referred to herein as an "RNAi agent". The double-stranded region of the RNAi agent may be 12 to 30 nucleotide pairs in length. For example, the double-stranded region may be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, 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 overhang may be 1 to 6 nucleotides in length, such as 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. The overhang may result from one strand being longer than the other, or from two strands of the same length being in a twisted shape. The overhang may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be another sequence. The first and second strands may also be linked, for example, by additional bases to form a hairpin, or by other non-base linkers.

[0181] In one embodiment, each nucleotide in the overhang region of the RNAi agent is independently a modified or unmodified nucleotide, such as, but not limited to, a 2'-sugar modification, such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT may be an overhang sequence at either end on either strand. The overhang may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be another sequence.

[0182] The 5'- or 3'-overhangs 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 having a phosphorothioate between two nucleotides, where the two nucleotides may 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, this 3'-overhang is present within the antisense strand. In one embodiment, this 3'-overhang is present within the sense strand.

[0183] The RNAi agent may contain only a single overhang that 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. The RNAi may also have blunt ends 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 a blunt 5' end. Without being bound by theory, in the case of an asymmetric blunt end at the 5' end of the antisense strand and an overhang at the 3' end of the antisense strand, it is preferred to load the guide strand into the RISC process.

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

[0185] When a 2-nucleotide overhang is present at the 3'-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three nucleotide ends, where two of the three nucleotides are overhang nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages 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 and antisense strands of the RNAi agent, including nucleotides that are part of the motif, are modified nucleotides. In one embodiment, each residue is independently modified, for example in an alternating motif, with 2'-O-methyl or 3'-fluoro. In one embodiment, all of the nucleotides of the iRNA of the invention are modified and the iRNA comprises 2'-fluoro modifications of 8 or less (e.g., 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less) on the sense strand and 2'-fluoro modifications of 6 or less (e.g., 5 or less, 4 or less, 3 or less, or 2 or less) on the antisense strand. 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 on three consecutive nucleotides, where one of the motifs is present at the cleavage site within the sense strand.

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

[0188] In an RNAi agent having a double-stranded region 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically at positions about 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs may be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, where the count starts from the first nucleotide from the 5' end of the antisense strand or the count starts 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 from the 5' end.

[0189] The sense strand of the RNAi agent may have at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; also, the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be aligned such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif within the sense strand forms a base pair with at least one of the three nucleotides of the motif within the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0190] In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including some nucleotides of the motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more alterations of one or both of the non-bridging phosphate oxygen and / or one or more bridging phosphate oxygens; alterations of the ribose sugar moiety, such as the 2'-hydroxyl of the ribose sugar; substantial substitution with "dephospho" linkers in the phosphate moiety; modification or substitution of the natural base; and substitution or modification of the ribose-phosphate backbone.

[0191] Since nucleic acids are polymers of subunits, many modifications, such as modifications of bases, or of the phosphate moiety, or of non-bridging O of the phosphate moiety, occur at positions that are repeated within the nucleic acid. In some cases, the modification will occur at any targeted position in the nucleic acid, but often it will not. By way of example, the modification may occur only at the 3' or 5' terminal positions, or only within the terminal region, such as at a position on the terminal nucleotide or within the last 2, 3, 4, 5, or 10 nucleotides of the strand. The modification may occur in double-stranded regions, single-stranded regions, or both. The modification may occur only within the double-stranded region of the RNA, or only within the single-stranded region of the RNA. For example, phosphorothioate modification at the non-bridging O position may occur only at one or both ends, or only within the terminal region, such as at a position on the terminal nucleotide or within the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may occur within both double-stranded and single-stranded regions, particularly at the ends. The 5' end may be phosphorylated.

[0192] For example, it may be possible to enhance stability, include specific bases during overhangs, or include modified nucleotides or nucleotide substitutes in single-stranded overhangs, such as 5' or 3' overhangs, 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, by the modifications described herein. Modifications can include, for example, the use of modifications known in the art at the 2'-position of the ribose sugar, such as the use of deoxyribonucleotides in place of ribonucleotides of the nucleobase, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications, and modifications at the phosphate group, such as phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0193] In one embodiment, each residue of the sense strand and the antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. Those strands can include two or more modifications. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

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

[0195] In one embodiment, N a and / or N bincludes modifications of an alternating pattern. The term "alternating motif", as used herein, refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. Alternating nucleotides may refer to every other nucleotide, every third nucleotide, or a similar pattern. For example, if each of A, B, and C represents one type of modification to a nucleotide, an alternating motif may be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…", or "ABCABCABCABC…", etc.

[0196] The types of modifications included within an alternating motif may be the same or different. For example, if each of A, B, C, and D represents one type of modification to a nucleotide, the alternating pattern, i.e., the modification to every other nucleotide, may be the same, but each of the sense strand or the antisense strand may be selected from several modification possibilities within the alternating motif, such as "ABABAB…", "ACACAC…", "BDBDBD…", or "CDCDCD…", etc.

[0197] In one embodiment, the RNAi agent of the present invention involves that the modification pattern in the alternating motif on the sense strand changes with respect to the modification pattern in the alternating motif on the antisense strand. It may be a change such that the modified group of the nucleotide on the sense strand corresponds to a different modified group of the nucleotide on the antisense strand, and vice versa. For example, when the sense strand pairs with the antisense strand in the dsRNA duplex, within the duplex region, the alternating motif within the sense strand may start with "ABABAB" from the 5'-3' of the strand, and the alternating motif within the antisense strand may start with "BABABA" from the 5'-3' of the strand. As another example, within the duplex region, the alternating motif within the sense strand may start with "AABBAABB" from the 5'-3' of the strand, and the alternating motif within the antisense strand may start with "BBAABBAA" from the 5'-3' of the strand, thereby resulting in a complete or partial change in the modification pattern between the sense strand and the antisense strand.

[0198] In one embodiment, the RNAi agent involves that the pattern of the alternating motif of 2'-O-methyl modification and 2'-F modification on the first sense strand has a change with respect to the pattern of the alternating motif of 2'-O-methyl modification and 2'-F modification on the first antisense strand, that is, the 2'-O-methyl modified nucleotide on the sense strand base pairs with the 2'-F modified nucleotide on the antisense strand, and vice versa. The 1st position of the sense strand may start with a 2'-F modification, and the 1st position of the antisense strand may start with a 2'-O-methyl modification.

[0199] By introducing one or more motifs of three identical modifications onto three consecutive nucleotides of the sense strand and / or the antisense strand, the initial modification pattern existing within the sense strand and / or the antisense strand is disrupted. This disruption of the modification pattern of the sense and / or antisense strands by introducing one or more motifs of three identical modifications onto three consecutive nucleotides of the sense strand and / or the antisense strand unexpectedly enhances the gene silencing activity against the target gene.

[0200] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotides adjacent to the motif is a modification different from the modification of the motif. For example, a portion of the sequence containing the motif is “…N a YYYN b …”, where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotides, and “N a ” and “N b ” represent the modifications to the nucleotides adjacent to the motif “YYY” that are different from the modification of Y, and where N a and N b can be the same or different modifications. Alternatively, N a and / or N b may or may not be present when a wing modification is present.

[0201] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The internucleotide linkage modification of phosphorothioate or methylphosphonate may occur on any nucleotide at any position of the strand, either on the sense strand or the antisense strand or both strands. For example, the internucleotide linkage modification may occur on all nucleotides 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 as or different from that on the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may also 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 includes internucleotide linkage modifications of phosphorothioate or methylphosphonate within the overhang region. For example, the overhang region may include two nucleotides having an internucleotide linkage of phosphorothioate or methylphosphonate between two nucleotides. The internucleotide linkage modification may also be provided to link the overhang nucleotide to the paired nucleotide at the end within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked through an internucleotide linkage of phosphorothioate or methylphosphonate, and optionally, there may be additional internucleotide linkages of phosphorothioate or methylphosphonate that link the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two internucleotide linkages of phosphorothioate between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third is a paired nucleotide adjacent to the overhang nucleotide. These three terminal nucleotides may be present 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, the two-nucleotide overhang is present at the 3' end of the antisense strand and there are two internucleotide linkages of phosphorothioate between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third is a paired nucleotide adjacent to the overhang nucleotide. Optionally, the RNAi agent may further have two internucleotide linkages of phosphorothioate between the three terminal 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 double strand, or in combinations thereof. The mismatches may be present within the overhang region or within the double strand region. Base pairs may be ranked based on their tendency to promote dissociation or melting (e.g., the simplest approach is to consider the pairing in terms of the free energy of association or dissociation of a particular pairing, based on individual pairings, but closely related or similar analyses can also be used). From the perspective of promoting dissociation, A:U is more preferred than G:C; G:U is more preferred than G:C; and I:C is more preferred than G:C (I = inosine). Mismatches, such as non-canonical or off-canonical pairings (as described elsewhere in this specification), are more preferred than canonical (A:T, A:U, G:C) pairings; also, pairings containing universal bases are more preferred than canonical pairings.

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

[0206] In one embodiment, the nucleotide at the 1st position inside the double strand region from the 5' end in 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 inside the double strand region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair inside the double strand region from the 5' end of the antisense strand is an AU base pair.

[0207] In one embodiment, the sense strand sequence has the formula (I): 5’n p -N a -(XXX)i-N b -YYY-N b -(ZZZ) j -N a -n q 3’(I) (wherein i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a represents an oligonucleotide sequence containing modified nucleotides from 0 to 25, and each sequence contains at least two different modified nucleotides; each N b represents an oligonucleotide sequence containing modified nucleotides from 0 to 10; each n p and n q each independently represents an overhang nucleotide; 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) may be represented by. Preferably, YYY is all 2'-F modified nucleotides.

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

[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 17 to 23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., it can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), where the count starts from the first nucleotide at the 5' end or, optionally, the count 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 both i and j are 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) may 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. Each N a may independently represent an oligonucleotide sequence containing 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. Each N a may independently represent an oligonucleotide sequence containing 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 independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X, Y and Z may be the same as or different from 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) and may be represented by:

[0215] When the sense strand is represented by formula (Ia), each N a may independently represent an oligonucleotide sequence comprising from 2 to 20, from 2 to 15, or from 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) (wherein k and l are each independently 0 or 1; p’ and q’ are each independently from 0 to 6; each N a ’ independently represents an oligonucleotide sequence comprising from 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides; each N b ’ independently represents an oligonucleotide sequence comprising from 0 to 10 modified nucleotides; each n p ’ and n q ’ independently represent overhang nucleotides; wherein 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) and may be represented by:

[0217] In one embodiment, N a ’ and / or N b ’ includes the modification of an alternating pattern.

[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 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 the count starts from the first nucleotide at the 5’ end or, optionally, the count starts from the first paired nucleotide within the double-stranded region at the 5’ end. Preferably, the Y’Y’Y’ motif occurs at positions 11, 12, 13.

[0219] In one embodiment, the Y’Y’Y’ motif consists of 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) and can be represented by.

[0222] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0223] When the antisense strand is represented by formula (IIc), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0224] When the antisense strand is represented by formula (IId), each N b ’ independently represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10. Preferably, N 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) and may be represented by.

[0226] When the antisense strand is represented as formula (IIa), each N a' represents an oligonucleotide sequence independently comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X', Y', and Z' may be the same as or different from one another. Each nucleotide of the sense strand and the antisense strand 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 of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. Each of X, Y, Z, X', Y', and Z' may particularly 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, and the count starts from the first nucleotide at the 5'-end or, optionally, the count starts from the first paired nucleotide inside the double-stranded region from the 5'-end, and Y represents a 2'-F modification.

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

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

[0230] Thus, the RNAi agent used in the method of the present invention may contain a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double strand has the formula (III): Sense: 5'n p -Na -(XXX)i-N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’ (III) (wherein i, j, k, and l are each independently 0 or 1; p, p’, q, and q’ are each independently 0 to 6; each N a and N a ’ represents an oligonucleotide sequence containing modified nucleotides from 0 to 25, and each sequence contains at least two different modified nucleotides; each N b and N b ’ represents an oligonucleotide sequence containing modified nucleotides from 0 to 10; where each n p ’, n p , n q ’, and n q may each be present or absent and independently represent overhang nucleotides; 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 represented 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 both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0232] Exemplary combinations of sense and antisense strands forming an RNAi double strand are represented by 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) contains

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

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

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

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

[0237] When the RNAi agent is represented by formula (IIIe), each N a , N a ’, Nb, and N b ’ represents an oligonucleotide sequence containing 0 to 25 nucleotides which are independently either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides.

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

[0239] When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), at least one of the Y nucleotides may form a base pair with one of the Y’ nucleotides. Alternatively, at least two of the Y nucleotides may form base pairs with the corresponding Y’ nucleotides; or all three of the Y nucleotides may form base pairs with the corresponding Y’ nucleotides.

[0240] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z’ nucleotides. Alternatively, at least two of the Z nucleotides may form base pairs with the corresponding Z’ nucleotides; or all three of the Z nucleotides may form base pairs with the corresponding Z’ nucleotides.

[0241] When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X’ nucleotides. Alternatively, at least two of the X nucleotides may form base pairs with the corresponding X’ nucleotides; or all three of the X nucleotides may form base pairs with the corresponding X’ nucleotides.

[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 ' is linked to an adjacent nucleotide via a phosphorothioate bond. 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 ' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described below). 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 ' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is linked to one or more GalNAc derivatives linked via a divalent 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 2'-fluoro modification and n p '>0, and at least one n p' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond and is linked to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0245] In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe) are linked to each other at one or both of the 5'-end and the 3'-end and optionally linked 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. Such publications include WO 2007 / 091269 pamphlet, US Patent No. 7,858,769 specification, WO 2010 / 141511 pamphlet, WO 2007 / 117686 pamphlet, WO 2009 / 014887 pamphlet and WO 2011 / 031520 pamphlet (the entire contents of each of which are hereby incorporated by reference herein).

[0247] As described in further detail below, an RNAi agent comprising the attachment of one or more carbohydrate moieties to the RNAi agent can optimize one or more properties of the RNAi agent. Often, 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 another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is so replaced is referred to herein as a ribose replacement modified subunit (RRMS). The cyclic carrier may 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 may be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system or may include two or more rings, such as fused rings. The cyclic carrier may be a fully saturated ring system or may include one or more double bonds.

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

[0249] The RNAi agent may be bound to the ligand via a carrier, where the carrier can be a cyclic group or an 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, the RNAi agent used in the method of the present 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 contain 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 present invention involves chemically linking one or more ligands, moieties or complexes to the RNA 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., Biorg. Med. Chem. Let., 1994, 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocolesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids such as 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); palmitoyl moieties (Mishra et al., Biochim. Biophys.Acta, 1995, 1264: 229-237); or octadecylamine or hexylamino-carbonyloxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: 923-937), but are not limited thereto.

[0253] In one embodiment, the ligand alters the distribution, targeting or lifespan of the iRNA agent in which it is incorporated. In a preferred embodiment, the ligand provides an improved affinity for a selected target, such as a molecule, cell or cell type, such as a compartment, such as an intracellular or intra-organ compartment, a body tissue or organ or region, for example, compared to a species in which such a ligand is absent. Preferred ligands do not participate in duplex pairing formation in double-stranded nucleic acids.

[0254] The ligand may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer like a synthetic polyamino acid. Examples of polyamino acids include polyamino acids, polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, peptidomimetic-polyamine, peptide-mimicking polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine quaternary salt, or α-helical peptide.

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

[0256] Other examples of ligands include dyes, intercalators (e.g., acridine), cross-linking agents (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) lithocholic acid, O3-(oleoyl) cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, aminos, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamines, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole complex, Eu3+ complex of tetraaza macrocyclic compound), dinitrophenyl, HRP, or AP.

[0257] The ligand can be a protein such as a glycoprotein; or a peptide such as a molecule having specific affinity for a co-ligand; or an antibody such as an antibody that binds to a specified cell type such as a liver cell. The ligand may also include hormones and hormone receptors. They may also include non-peptide chemical species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, or polyvalent fucose. The ligand can be, for example, a lipopolysaccharide, a p38 MAP kinase activator, or an NF-κB activator.

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

[0259] In some embodiments, the ligand that attaches to the iRNA described herein refers to a pharmacokinetic modulator (PK modulator). Examples of PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like, but are not limited thereto. Oligonucleotides containing some phosphorothioate linkages are also known to bind to serum proteins, and thus, for example, short-chain oligonucleotides such as about 5-base, 10-base, 15-base, or 20-base oligonucleotides containing a plurality of phosphorothioate linkages in the backbone are also suitable for use in the present invention as ligands (e.g., as PK modulating ligands). 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] The ligand-conjugated oligonucleotides of the present invention may be synthesized by the use of oligonucleotides having pendant reactive functional groups, such as those derived from the addition of a binding molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with a commercially available ligand, a synthesized ligand having any of a variety of protecting groups, or a ligand having a binding moiety to be attached.

[0261] The oligonucleotides used in the complexes of the present invention may advantageously and conventionally be generated through well-known solid-phase synthesis techniques. Apparatuses for such synthesis are sold by several suppliers, including Applied Biosystems (Foster City, Calif.). In addition or as an alternative, any other means for such synthesis known in the art may be used. It is also known to prepare other oligonucleotides, such as phosphorothioate and alkylated derivatives, using similar techniques.

[0262] In the ligand-conjugated oligonucleotides of the present invention, and sequence-specific binding nucleosides carrying ligand molecules, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside complex precursors already carrying a linking moiety, ligand-nucleotide or nucleoside-complex precursors already carrying a ligand molecule, or basic units carrying a non-nucleoside ligand.

[0263] When using nucleotide complex precursors already having a linking moiety, the synthesis of the sequence-specific binding nucleoside is typically completed, and then the ligand molecule is reacted with the linking moiety to generate the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or binding nucleosides of the present invention are commercially available and are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside complexes in addition to standard and non-standard phosphoramidites conventionally used in oligonucleotide synthesis.

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

[0265] For inhibition, lipid-based ligands can be used, for example, to control the binding of the complex to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and thus less likely to be removed from the body. Lipids or lipid-based ligands that bind more weakly to HSA can be used to target the complex to the kidney.

[0266] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity such that the complex is preferably distributed to non-renal tissues. However, the affinity is preferably not so strong that the 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 complex is preferably distributed to the kidney. Other moieties that target renal cells can also be used instead of or in addition to the lipid-based ligand.

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

[0269] B. Cell Penetrating Agents In another aspect, the ligand is a cell penetrating agent, preferably a helical cell penetrating agent. Preferably, the cell penetrating agent is amphiphilic. Exemplary cell penetrating agents are peptides such as tat or antennopedia. When the cell penetrating agent is a peptide, it can be modified, including peptidomimetics, reverse isomers, non-peptides or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having both lipophilic and hydrophobic phases.

[0270] The ligand can be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to a natural peptide. The addition of peptides and peptidomimetics to the iRNA agent can affect the pharmacokinetic distribution of the iRNA, such as by promoting cell recognition and uptake. The peptide or peptidomimetic moiety can be, for example, about 5 to 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 amphiphilic peptide, or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, a constrained peptide or a cross-linked peptide. In another alternative, the peptide moiety can contain a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having 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 target moiety. The peptide moiety can be a "delivery" peptide that can transport a number of polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. 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 found to be able to function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or a 1-bead 1-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of peptides or peptidomimetics tethered to the dsRNA agent through incorporated monomer units for cell targeting purposes are arginine-glycine-aspartic acid (RGD)-peptides, or RGD mimetics. The peptide moiety can range from about 5 amino acids to about 40 amino acids in length. The peptide moiety can have structural modifications such as to increase stability or to induce conformational properties. Any of the structural modifications described below can be used.

[0272] The RGD peptides used in the compositions and methods of the present invention may be linear or cyclic, and may be modified, for example, by glycosylation or methylation to facilitate targeting to a particular tissue. RGD-containing peptides and peptidomimetics include D-amino acids, as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands may be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0273] A "cell-permeable peptide" can penetrate cells such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-permeable peptides can be, for example, α-helical linear peptides (such as LL-37 or Ceropin P1), disulfide bond-containing peptides (such as α-defensin, β-defensin or bacteriocin), or peptides containing only one or two major amino acids (such as PR-39 or indolicidin). The cell-permeable peptide may also contain a nuclear localization signal (NLS). For example, the cell-permeable peptide can be a bipartite amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0274] C. Carbohydrate Conjugates In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a carbohydrate itself composed of one or more monosaccharide units having at least 6 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 as a part thereof a carbohydrate moiety composed of one or more monosaccharide units each having at least 6 carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include saccharides (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 saccharides with TTR or higher (e.g., TTR, C6, C7, or C8); disaccharides and trisaccharides include saccharides having 2 or 3 monosaccharide units (e.g., TTR, C6, C7, or C8).

[0275] In one embodiment, the carbohydrate complex used in the compositions and methods of the present invention is a monosaccharide. In another embodiment, the carbohydrate complex used in the compositions and methods of the present invention is selected from the group consisting of

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0276] In one embodiment, the monosaccharide is N-acetylgalactosamine, for example,

Chemical formula

[0277] Another exemplary carbohydrate conjugate used in the embodiments described herein includes, but is not limited to,

Chemical formula

[0278] In certain embodiments of the invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the invention via a trivalent linker.

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

[0280] In some embodiments, for example, when the two strands of the iRNA agent of the invention form a hairpin loop containing a plurality of unpaired nucleotides and are part of one larger molecule connected by an uninterrupted strand of nucleotides between the 3' end of one strand and the 5' end of each other strand, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in 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 modifier and / or a cell penetrating peptide.

[0282] Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publication 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 can be attached to the iRNA oligonucleotide by various linkers that can be cleavable or non-cleavable.

[0284] The term "linker" or "linking group" means an organic moiety that connects two moieties of a compound, such as by covalent bonding to two moieties of the compound. A linker typically contains a chain of atoms, such as a direct bond, or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or 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, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, but not limited thereto, and one or more of its methylenes are O, S, S(O), SO2, N(R 8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, wherein R8 can be interrupted or terminated by (which is hydrogen, acyl, aliphatic or substituted aliphatic). In one embodiment, the linker is about 1 to 24 atoms, 2 to 24 atoms, 3 to 24 atoms, 4 to 24 atoms, 5 to 24 atoms, 6 to 24 atoms, 6 to 18 atoms, 7 to 18 atoms, 7 to 17 atoms, 8 to 17 atoms, 6 to 16 atoms, 7 to 16 atoms, or 8 to 16 atoms.

[0285] The cleavable linking group is sufficiently stable extracellularly, but is cleaved upon entry into the target cell to release the two moieties that the linker is connecting. In a preferred embodiment, the cleavable linking group 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 a first standard condition (e.g., selected to mimic or correspond to intracellular conditions), in the blood of the subject, or under a second standard condition (e.g., selected to mimic or correspond to conditions found in blood or serum).

[0286] The cleavable linking group is susceptible to the influence of cleavage agents such as, for example, pH, redox potential or the presence of degradable molecules. Generally, cleavage agents are more common or are found at higher levels or activities in cells than in serum or blood. Examples of such degradable agents include, for example, redox agents selected for or without substrate specificity, such as oxidoreductases or reducing agents such as mercaptans present in cells that can decompose redox-cleavable linking groups by reduction; esterases; agents that can provide an acidic environment such as endosomes or those that provide a pH of 5 or less; general acids, peptidases (which can be substrate specific), and enzymes that can hydrolyze or decompose acid-cleavable linking groups by acting as phosphatases.

[0287] Cleavable linkers 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 in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linkages that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand in the cell or into a desired compartment of the cell.

[0288] The linker can include a cleavable linkage that is cleavable by a specific enzyme. The type of cleavable linkage incorporated into the linker can depend on the targeted cell. For example, a ligand targeting the liver can be linked to the cationic lipid through a linker containing an ester group. Hepatocytes are rich in esterases, and thus the linker is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

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

[0290] In general, the suitability of a candidate cleavable linking group can be evaluated by examining the ability of a degrading agent (condition) to cleave the candidate linking group. It is also desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, a relative sensitivity of cleavage between a first condition selected to show cleavage in target cells and a second condition selected to show cleavage in other tissues or in a biological fluid such as, for example, blood or serum can be determined. The evaluation can be carried out in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may also be useful to perform an initial evaluation under cell-free or culture conditions and confirm by further evaluation in a whole animal. In a preferred embodiment, a useful candidate compound is 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 linking group In one embodiment, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-S-S-). To determine whether a cleavable linking group candidate is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular target agent, one can rely on the methods described herein. For example, a candidate 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. A candidate can also be evaluated under conditions selected to mimic blood or serum conditions. One candidate compound is cleaved up to about 10% in blood. In other embodiments, a useful candidate compound is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90 or about 100 times more rapidly degraded 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 cleavage rate of a candidate compound can be determined using a standard enzyme kinetics assay under conditions selected to mimic intracellular media, compared to conditions selected to mimic extracellular media.

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

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

[0294] iv. Ester-based linking group In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases inside a cell. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using a method similar to that described above.

[0295] v. Peptide-based cleavage group In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved intracellularly by enzymes such as peptidases and proteases. The peptide-based cleavable linking group is a peptide bond formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelene. The peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. The peptide-based cleaving group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linking group generally has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0296] In one embodiment, the iRNA of the present invention conjugates with a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrates conjugated with the linker of the compositions and methods of the present invention include

Chemical formula

Chemical formula

[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 divalent or trivalent branched linker.

[0298] In one embodiment, the dsRNA of the present invention Formulas (XXXII) to (XXXV),

Chem.

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

[0300] Representative U.S. patents that teach the preparation of RNA complexes, each of which is hereby incorporated herein by reference in its entirety, are U.S. Patent No. 4,828,979; U.S. Patent No. 4,948,882; U.S. Patent No. 5,218,105; U.S. Patent No. 5,525,465; U.S. Patent No. 5,541,313; U.S. Patent No. 5,545,730; U.S. Patent No. 5,552,538; U.S. Patent No. 5,578,717; U.S. Patent No. 5,580,731; U.S. Patent No. 5,591,584; U.S. Patent No. 5,109,124; U.S. Patent No. 5,118,802; U.S. Patent No. 5,138,045; U.S. Patent No. 5,414,077; U.S. Patent No. 5,486,603; U.S. Patent No. 5,512,439; U.S. Patent No. 5,578,718; U.S. Patent No. 5,608,046; U.S. Patent No. 4,587,044; U.S. Patent No. 4,605,735; U.S. Patent No. 4,667,025; U.S. Patent No. 4,762,779; U.S. Patent No. 4,789,737; U.S. Patent No. 4,824,941; U.S. Patent No. 4,835,263; U.S. Patent No. 4,876,335; U.S. Patent No. 4,904,582; U.S. Patent No. 4,958,013; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,245,022; U.S. Patent No. 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;U.S. Patent No. 5,567,810; U.S. Patent No. 5,574,142; U.S. Patent No. 5,585,481; U.S. Patent No. 5,587,371; U.S. Patent No. 5,595,726; U.S. Patent No. 5,597,696; U.S. Patent No. 5,599,923; U.S. Patent Nos. 5,599,928 and 5,688,941; U.S. Patent No. 6,294,664; U.S. Patent No. 6,320,017; U.S. Patent No. 6,576,752; U.S. Patent No. 6,783,931; U.S. Patent No. 6,900,297; U.S. Patent No. 7,037,646; U.S. Patent No. 8,106,022, but are not limited thereto.;

[0301] Not all positions in a given compound need to be uniformly modified, and in fact, two or more of the foregoing modifications can be incorporated in a single compound or even in a single nucleoside within an iRNA. The invention also includes iRNA compounds that are chimeric compounds.;

[0302] "Chimeric" iRNA compounds or "chimeras" are, in the context of the present invention, iRNA compounds, preferably dsRNA, containing 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 is modified so as to confer upon the iRNA an increased resistance to nuclease degradation, an increased cellular uptake, and / or an increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as an enzyme substrate capable of cleaving an RNA:DNA or RNA:RNA hybrid. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly enhancing the iRNA inhibition efficiency of gene expression. As a result, when chimeric dsRNA is used, comparable results are often obtained with shorter iRNAs as compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, by related nucleic acid hybridization techniques known in the art.

[0303] In some cases, the RNA of the iRNA can be modified by non-ligand groups. To enhance the activity, cellular distribution or cellular uptake of the iRNA, several non-ligand molecules are conjugated to the iRNA, and procedures for performing such conjugation are available in the academic 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. N.Y. Acad. Sci., 1992, 660: 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3: 2765), thiocolesterol (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), palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229), or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (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 having an amino linker at one or more positions of the sequence. Appropriate coupling or activating reagents are then used to react the amino group with a molecule to which it is to be conjugated. The conjugation reaction can be carried out in solution phase, while the RNA remains bound to a solid support, or following RNA cleavage. RNA complex purification by HPLC typically gives a pure complex.

[0304] V. Delivery of the Inventive iRNA Delivery of the inventive iRNA to cells, such as cells within a subject, e.g., a human subject (such as a subject in need thereof, e.g., a subject having a disease, disorder or condition associated with TTR), can be accomplished in several different ways. For example, delivery may be effected by contacting the cells with the inventive iRNA, either in vitro or in vivo. In vivo delivery may also be effected directly, for example, by administering to the subject a composition comprising an iRNA, such as dsRNA. As an alternative, in vivo delivery may be effected indirectly by administering one or more vectors that encode and induce expression of the iRNA. These alternatives are considered 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 WO 94 / 02595, which are incorporated herein by reference in their entireties). In in vivo delivery, factors to be considered for delivering iRNA molecules include, for example, the biological stability of the delivery molecule, prevention of non-specific effects, and accumulation of the delivery molecule in the target tissue. Non-specific effects of iRNA can be minimized, for example, by local administration such as direct injection or implantation into tissue or topical administration of a formulation. Local administration to the site of treatment maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be damaged or degraded by the agent, and allows administration at a lower total dose of the iRNA molecule. Several studies have shown successful gene product knockdown when iRNA is administered locally. For example, intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138), and subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216), both showed prevention of neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice decreased tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and could extend the survival of mice bearing tumors (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523).RNA interference has shown success in local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P.H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G.T., et al (2004) Neuroscience 129:521-528; Thakker, E.R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602), and to the lung by intranasal administration (Howard, K.A., et al (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). To systemically administer iRNA for treating diseases, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of dsRNA by endogenous and exo-nucleases in vivo. Modification of the RNA or pharmaceutical carrier can also enable targeting of the iRNA composition to the target tissue, avoiding unwanted non-specific effects. Chemical conjugation of a lipophilic group such as cholesterol to the iRNA molecule can enhance cellular uptake and prevent degradation. For example, an iRNA that counteracts ApoB conjugated to a lipophilic cholesterol moiety was systemically injected into mice, resulting in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an aptamer to iRNA has been shown to suppress tumor growth and mediate tumor regression in a mouse model of prostate cancer. (McNamara, J.O., et al (2006) Nat. Biotechnol. 24:1005-1015).In an embodiment of the alternative, the iRNA can be delivered using a drug delivery system such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. The positively charged cationic delivery system facilitates the binding of the iRNA molecule (negatively charged) and also enhances the interaction with the negatively charged cell membrane, enabling efficient uptake of the iRNA by the cell. Cationic lipids, dendrimers, or polymers can be conjugated to the iRNA or induced to form vesicles or micelles that encapsulate the iRNA (see, for example, Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of the iRNA upon systemic administration. Methods for creating and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are hereby incorporated 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 (2008) Pharm. Res. Aug 16 online first; 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 forms a complex with cyclodextrin. The methods of administration and pharmaceutical compositions of iRNA and cyclodextrin are in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0306] A. Vector encoding the iRNA of the present invention TTR gene-targeted iRNA can be expressed from a transcription unit inserted into a DNA or RNA vector (see, for example, Couture, A., et al., TIG. (1996), 12:5-10; Skillern, A., et al., International Publication No. 00 / 22113 pamphlet; Conrad, International Publication No. 00 / 22114 pamphlet; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from several hours to about several weeks) or persistent (from several weeks to several months or more), depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. The transgene can also be constructed to allow it to be inherited as an episomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

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

[0308] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for iRNA expression described herein can be generated using expression vectors that are compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial suppliers. Typically, such vectors are provided with convenient restriction enzyme recognition sites for insertion of the desired nucleic acid fragment. Delivery of the iRNA expression vector can be by systemic administration, such as intravenous or intramuscular administration, administration to target cells explanted from the patient followed by reintroduction into the patient, or any other means that allows introduction into the 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 that target different regions of the target RNA over a period of more than one week are also contemplated by the present invention. Successful introduction of the vector into the host cell can be monitored using a variety of 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 by using markers that provide resistance to specific environmental factors (e.g., antibiotics and drugs) to the transfected cells, such as hygromycin B resistance.

[0310] Viral vector systems that can be used with the methods and compositions described herein include: (a) adenoviral vectors; (b) retroviral vectors such as, but not limited to, lentiviral vectors, Moloney murine leukemia virus; (c) adeno-associated viral 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, but are not limited thereto. Replication-deficient viruses may also be advantageous. Different vectors may or may not integrate into the genome of the cell. The construct may, if desired, contain viral sequences for transfection. Alternatively, the construct may be incorporated into a vector capable of episomal replication, such as, for example, EPV and EBV vectors. Constructs for the recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure iRNA expression in the target cell. Other aspects considered for the vectors and constructs are described in more detail below.

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

[0312] The expression of iRNA can be precisely regulated, for example, using inducible control 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 the control of 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). One of ordinary skill in the art can select an appropriate regulatory / promoter sequence based on the intended use of the iRNA transgene.

[0313] Viral vectors containing nucleic acid sequences encoding iRNA 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 sequence encoding iRNA is cloned into one or more vectors that facilitate delivery of the nucleic acid to the patient. For more details on retroviral vectors, see, for example, Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells to generate stem cells with high resistance to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are 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 whose use is contemplated include, for example, the 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 considered for use in the delivery of the iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes, for example, to airway epithelia. Adenoviruses naturally infect airway epithelia and cause mild disease. Other targets for adenovirus-based delivery systems are the liver, the 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 an adenovirus vector for transferring genes to the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy are 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 pamphlet; and Wang, et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing the iRNAs addressed in the present invention, methods of constructing recombinant AV vectors, and methods of 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 iRNA of the present invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA addressed in the present invention, methods of constructing recombinant AV vectors, and methods of delivering the vectors to target cells are incorporated herein by reference in their entirety: Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J 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; International Publication No. 94 / 13788 Pamphlet; and International Publication No. 93 / 24641 Pamphlet.

[0316] Another viral vector suitable for delivering the iRNA of the present invention is, for example, a vaccinia virus such as attenuated vaccinia like modified virus Ankara (MVA) or NYVAC, or a poxvirus such as avipox like fowlpox or canarypox.

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

[0318] The pharmaceutical of the vector can contain the vector in an acceptable diluent, or can contain a sustained-release matrix in which the gene delivery vehicle is encapsulated. As an alternative, for example, if a complete gene delivery vector such as a retroviral vector can be produced intact from recombinant cells, the pharmaceutical can contain one or more cells that produce the gene delivery system.

[0319] VI. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations containing the iRNA of the present invention. In one embodiment, provided by the present invention is a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition containing iRNA is useful for treating a disease or disorder associated with the expression or activity of the TTR gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery, such as subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma by intracerebral infusion, such as continuous pump infusion. The pharmaceutical composition of the present invention may be administered in a dose sufficient to inhibit the expression of the TTR gene. In one embodiment, the iRNA agent of the present invention, such as a dsRNA agent, 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, over a period of 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. The administration can be repeated, for example, regularly, such as weekly, bi-weekly (i.e., every two weeks), for 1 month, 2 months, 3 months, 4 months or longer. The administration can also be repeated, for example, on a monthly basis, or on a quarterly basis, for example, every about 12 weeks. After the initial treatment plan, the therapeutic agent can be administered at a lower frequency basis. For example, after administration weekly or bi-weekly for 3 months, the administration can be repeated monthly for 6 months or more than 1 year.

[0321] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as 2, 3 or more partial doses at appropriate intervals throughout the day, or even delivered through continuous infusion or controlled release formulations. In that case, the iRNA contained in each partial dose must correspondingly be less in order to achieve the total daily dose. The dosage unit can also be formulated for delivery over several days using, for example, conventional sustained release formulations that provide a sustained release of the iRNA over several days. Sustained release formulations are well known in the art and are particularly useful for the delivery of agents to specific sites, which can be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding plurality of daily doses.

[0322] In another embodiment, a single administration of the pharmaceutical composition can be over a long term such that subsequent doses are administered at intervals of 3, 4, or 5 days or less, at intervals of 1, 2, 3, or 4 weeks or less, or at intervals of 9, 10, 11, or 12 weeks or less. In some embodiments of the present invention, a single administration of the pharmaceutical composition of the present invention is administered once a week. In another embodiment of the present invention, a single administration 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 still other embodiments, a single dose of the pharmaceutical composition of the present invention is administered quarterly.

[0323] One of ordinary skill in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other existing diseases, can affect the dosage and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the composition can 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 can be estimated using conventional procedures or based on in vivo testing 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 region to be treated. Administration can be local (e.g., by transdermal patch), pulmonary by inhalation or insufflation of powders or aerosols, including but not limited to nebulizers; intratracheal, intranasal, transdermal and percutaneous, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal administration through, for example, an implant device; or intracranial administration, for example, into the brain parenchyma, subarachnoid space or ventricles of the brain.

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

[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, etc. may be necessary or desirable. Coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the iRNA addressed in the present invention is in admixture with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine DOPE, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA addressed in the present invention can be encapsulated within liposomes or can form a complex therewith, particularly with cationic liposomes. As an alternative, the iRNA can form a complex 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 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof), but are not limited thereto. Topical formulations are detailed 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 can be formulated for delivery within membrane-like molecular assemblies such as, for example, liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphipathic lipids arranged in at least one bilayer, such as, for example, one bilayer or multiple bilayers. Liposomes include monolayer or multilamellar vesicles having 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 the translocation and delivery of the active ingredient to the site of action. Since the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the liposomal bilayer fuses with the bilayer of the cell membrane. As the fusion of the liposome with the cell progresses, the internal aqueous contents containing the iRNA are delivered into the cell, where the iRNA can specifically bind to the target RNA and mediate the iRNA. In some cases, the liposomes are also specifically targeted, for example, to direct the iRNA to a particular cell type.

[0328] Liposomes containing the iRNA agent can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent such that micelles are formed in the absence of the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid complex. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles containing the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of the iRNA agent.

[0329] If desired, a carrier compound, for example, to assist condensation, can be added during the condensation reaction by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (such as spermine or spermidine). The pH can also be adjusted to assist condensation.

[0330] A method for generating a stable polynucleotide delivery vehicle that incorporates a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle is further described in WO 96 / 37194 pamphlet, which is hereby incorporated by reference in its entirety. Liposome formation may also include one or more aspects of the exemplary methods described in Felgner, P.L. et 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. M. Mol. Biol. 23:238, 1965; Olson, et 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. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication, and combinations of freeze-thawing and extrusion (see, for example, Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization may be used if consistently small (50-200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable for loading the iRNA agent preparations within liposomes.

[0331] Liposomes are classified 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 taken up inside endosomes. Due to the acidic pH within endosomes, the liposomes rupture and release 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 form complexes with nucleic acids; rather, they encapsulate it. Since both nucleic acids and lipids have similar charges, repulsion rather than complex formation occurs. Nevertheless, some nucleic acids are 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 foreign 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 derived phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, whereas anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (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] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent No. 5,283,185; U.S. Patent 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 liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied and their utility in drug delivery to the skin has been determined. Cyclosporin A was delivered intradermally into mouse skin using nonionic liposome formulations containing Novasome (trademark) I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome (trademark) II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results suggested that such nonionic liposome systems are effective in facilitating the deposition of cyclosporin A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0336] Liposomes also include "sterically stabilized" liposomes, which term as used herein refers to liposomes containing one or more specialized lipids which, when incorporated into the liposome, result in an improved circulation lifetime compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid moiety of the liposome is (A) monosialoganglioside G M1It contains one or more glycolipids such as, or (B) is derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, in the art, it is believed that for sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids, the improvement in the circulation half-life of these sterically stabilized liposomes is due to a reduced uptake by reticuloendothelial system (RES) cells (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. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactosylcerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924 pamphlet, both assigned to Allen et al., disclose liposomes containing (1) sphingomyelin and (2) ganglioside G M1 or galactosylcerebroside sulfate ester. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 pamphlet (Lim et al.).

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

[0339] Further advantages of liposomes include the following. Liposomes obtained from natural phospholipids are biocompatible and biodegradable; a wide range of water- and lipid-soluble agents can be incorporated into liposomes; and liposomes can protect encapsulated iRNA agents 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 the preparation of liposome formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0340] Small liposomes can be formed using N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a positively charged synthetic cationic lipid, which spontaneously interacts with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in iRNA agent delivery (for an explanation of the use of DOTMA in combination with its DNA, see, for example, Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355).

[0341] 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), an ADOTMA analog, can be used in combination with a phospholipid to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to cultured cells in vivo. It contains positively charged DOTMA liposomes that interact spontaneously with negatively charged polynucleotides to form complexes. By using sufficiently positively charged liposomes, the net charge on the resulting complex is also positive. The positively charged complex thus prepared adheres spontaneously to the negatively charged cell surface, fuses with the plasma membrane, and efficiently delivers functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonio)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are joined by ester rather than ether linkages.

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

[0343] Another cationic lipid complex contains lipid derivatization by cholesterol (「DC-Chol」) formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolyllysine generated 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 exhibit several advantages over other formulations. Such advantages include a reduction in side effects associated with high systemic absorption of the administered agent, an increased accumulation of the administered agent at the desired target, and the ability to administer iRNA agents into the skin. In some implementations, liposomes are used to deliver iRNA agents to epidermal cells and to enhance the penetration of iRNA agents into skin tissue, such as within the skin. For example, liposomes can be topically applied. The topical delivery of therapeutic agents formulated as liposomes to the skin has been demonstrated (see, for example, Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R.J. 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 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 and their utility in drug delivery to the skin has been determined. 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 intradermally into the skin of mice. Such formulations containing iRNA agents are useful for treating skin diseases.

[0346] Liposomes containing iRNA can be highly deformable. Such deformability can enable liposomes to penetrate through pores smaller than the average radius of the liposomes. For example, transferosomes are a type of deformable liposome. Transferosomes can be prepared by adding a surface edge activator, which is usually a surfactant, to a standard liposome composition. Transferosomes containing an iRNA agent can be delivered subcutaneously, for example, by infection, to deliver the iRNA agent to keratinocytes in 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. Further, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, often reach their targets without fragmentation, and often self-load.

[0347] Other formulations according to the present invention are described in PCT Publication WO 2008 / 042973, the entire content of which is incorporated herein by reference.

[0348] Transferosomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates as drug delivery vehicles. Transferosomes can be described as fat droplets that can easily penetrate through pores smaller than the droplets because they are very highly deformable. Transferosomes can adapt to the environment in which they are used. For example, they can self-optimize (adapt to skin pore shape), self-repair, often reach their targets without fragmentation, and often self-load. To prepare transferosomes, a surface edge activator, which is usually a surfactant, can be added to a standard liposome composition. Transferosomes have been used to deliver serum albumin to the skin. Transferosome-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 for classifying and grading the properties of a number of different surfactant types, both natural and synthetic, is by use of the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means of classifying the different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0350] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of uses in pharmaceuticals and cosmetic products and can be used over a wide pH range. Generally, their HLB values range from 2 to about 18 depending on their structure. Nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters are included in this class. 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 commonly seen members of the nonionic surfactant class.

[0351] When surfactant molecules carry a negative charge upon dissolution or dispersion in water, the surfactant is classified as anionic. Examples of anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, sulfate esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and acyl sulfosuccinates, and acyl phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0352] When surfactant molecules carry a positive charge upon dissolution or dispersion in water, the surfactant is classified as cationic. Examples of cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0353] When surfactant molecules have the ability to have either a positive or negative charge, the surfactant is classified as amphoteric. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids.

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

[0355] The iRNA used in the method of the present invention can also be provided as a micelle formulation. A "micelle" is defined herein as a particular type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such 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 reverse arrangement exists.

[0356] A mixed micelle formulation suitable for delivery through a transdermal membrane is an siRNA composition, an alkali metal C8 - C 22An aqueous solution of an alkyl sulfate and a micelle-forming compound may be mixed and prepared. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, lulitisa oil, evening primrose oil, menthol, trihydroxyoxocolanyl glycine and pharmaceutically acceptable salts thereof, 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 are formed by substantially mixing the components in any manner, but are vigorously mixed to provide smaller micelles.

[0357] In one method, a first micelle composition containing an siRNA composition and at least an alkali metal alkyl sulfate is prepared. Next, the first micelle composition is mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, the micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by the addition of 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 together with the micelle-forming components. An isotonic agent such as glycerin may also be added after the formation of the mixed micelle composition.

[0359] To deliver the micellar formulation as a spray, the formulation can be placed in a propellant metering dispenser and the dispenser can be loaded with a propellant. The propellant under pressure is in liquid form within the metering dispenser. The ratio of the components is adjusted such that the aqueous phase and the propellant phase become one, i.e., become a single phase. If there are two phases, for example, it is necessary to shake the metering dispenser before dispersing a portion of the contents through a metering valve. The dispensed dose of the pharmaceutical is injected as a fine spray from the metering valve.

[0360] Examples of 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 concentration of the essential components can be determined by relatively simple experimental methods. For absorption through the oral cavity, it is often desirable to increase the dose through injection, or for example, at least double or triple the dose administered through the gastrointestinal tract.

[0362] B. Lipid Particles For example, iRNA such as the dsRNA of the present invention may be formed by being completely encapsulated in a lipid formulation such as an LNP or other nucleic acid-lipid particles.

[0363] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents particle aggregation (e.g., a PEG-lipid conjugate). LNPs exhibit a long circulation lifetime following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically distant from the site of administration), making them highly useful for systemic applications. Examples of LNPs include "pSPLP" which includes an encapsulating condensing agent-nucleic acid complex described in WO 00 / 03683 pamphlet. 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 non-toxic. In addition, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is nuclease-resistant in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent No. 5,976,567; U.S. Patent No. 5,981,501; U.S. Patent No. 6,534,484; U.S. Patent No. 6,586,410; U.S. Patent No. 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and WO 96 / 40964 pamphlet.

[0364] In one embodiment, the ratio of lipid to drug (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 ranges cited above are also considered part of the present 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-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy(Dilinoleyoxy)-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy(Dilinoleyoxy)-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 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-dilinoleyl-oxy-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-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or an analog thereof, (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-tetraene-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 a mixture thereof. The cationic lipid may constitute about 20 mol% to about 50 mol% or about 40 mol% of the total lipids present in the particles.

[0366] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to create 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 (mole percentage), have a particle size of 63.0 ± 20 nm, and an siRNA / lipid ratio of 0.027.

[0368] The ionic / non-cationic lipid can be, but is not limited to, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a mixture thereof, and can also be an anionic lipid or a neutral lipid. When cholesterol is included, the non-cationic lipid can be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0369] The conjugate lipids that inhibit particle aggregation can be, for example, without limitation, polyethylene glycol (PEG)-lipids, such as PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA complex can be, for example, PEG-dilauroxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C18). The conjugate lipid that prevents particle aggregation can be from 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, for example, cholesterol in an amount of about 10 mol% to about 60 mol% or about 48 mol% of the total lipid present in the particles.

[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 Mar. 26, 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Each stock solution in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. Next, the stock solutions of ND98, cholesterol, and PEG-ceramide C16 can be combined, for example, at a molar ratio of 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. 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 a 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, such as 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.

Chemical formula

[0372] The LNP01 formulation is described in International Publication No. 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 pamphlet, filed on April 15, 2009, which is incorporated herein by reference.

[0378] Formulations containing XTC are described in WO 2010 / 088537 pamphlet (the entire content of which is incorporated herein by reference).

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

[0380] Formulations containing ALNY-100 are described in WO 2010 / 054406 pamphlet (the entire content of which is incorporated herein by reference).

[0381] Formulations containing C12-200 are described in WO 2010 / 129709 pamphlet (the entire content of which is incorporated herein by reference).

[0382] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or binders may be desirable. In some embodiments, the oral formulation is one in which the DsRNA addressed in the present invention is administered in combination with one or more penetration enhancing surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or their salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA), ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucuronic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, and sodium glycolidihydrofusidate. 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 a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, such as combinations of fatty acids / salts, for example in combination with bile acids / salts. One exemplary combination is the sodium salts of lauric acid, capric acid, and UDCA. Penetration enhancers further include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The DsRNA addressed in the present invention may be orally delivered in particulate form, including spray-dried particles, or may be complexed to form micro- or nanoparticles.Examples of dsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkyl cyanoacrylates; cationized gelatin, albumin, starch, acrylate, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylates; DEAE-derivatized polyimines, pullulans, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinyl pyridine, polythiodiethylaminomethyl ethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), poly(isohexyl cynaoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-co-glycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

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

[0384] Examples of the pharmaceutical composition of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of ingredients including, but not limited to, ready-made liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver disorders such as liver cancer, formulations targeting the liver are particularly preferred.

[0385] The pharmaceutical preparations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with a pharmaceutical carrier or excipient. In general, the preparations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier 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 which increase the viscosity of the suspension, such as, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspensions can also contain stabilizers.

[0387] C. Additional Formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. An emulsion is typically a heterogeneous system of one liquid dispersed in another liquid, usually in the form of droplets having a diameter greater than 0.1 μm (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, N.Y., volume 1, p. 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often two-phase systems that contain two immiscible liquid phases that are intimately mixed and dispersed in each other. Generally, an emulsion can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed in the bulk oil phase and dispersed as fine droplets, the resulting composition is referred to as a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely dispersed in the bulk aqueous phase and dispersed as fine droplets, the resulting composition is referred to as an oil-in-water (o / w) emulsion.An emulsion may contain additional components in addition to the dispersed phase, the active agent, which may exist as a solution in either the aqueous or the oily phase or as a separate phase itself. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as required. Pharmaceutical emulsions may also be multiple emulsions containing more than two phases, for example in the case of water-in-oil-in-water (o / w / o) and oil-in-water-in-oil (w / o / w) emulsions. Such complex formulations often offer certain advantages not provided by simple two-component emulsions. A multiple emulsion in which the individual oil droplets of an o / w emulsion enclose small water droplets constitutes a w / o / w emulsion. Similarly, an oil droplet system encapsulated in small spheres of water and stabilized within an oily continuous phase provides an o / w / o emulsion.

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

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

[0390] Examples of natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorbent bases such as anhydrous lanolin and hydrophilic petrolatum, which have hydrophilic properties such that they can absorb water to form w / o emulsions, still maintain their semi-solid viscosity. Finely divided solids are also used as excellent emulsifiers, especially in combination with surfactants, in viscous preparations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloids of aluminum silicate and 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. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, wetting agents, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0392] Examples of hydrophilic colloids or hydrocolloids include natural gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These are dispersed or swollen in water to form a colloidal solution that stabilizes the emulsion by forming a strong interfacial film around the 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 that can readily support the growth of microorganisms, so preservatives are often incorporated into these formulations. Commonly used preservatives included in emulsion formulations are methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also generally added to emulsion formulations to prevent degradation of the formulation. Antioxidants used can be free radical scavengers such as tocopherol, alkyl gallates, butylated hydroxyanisole, and 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 the dermal, oral, and parenteral routes, and methods for manufacturing them, have been reviewed in the literature. (See, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich, N.G., and Ansel, H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery are very widely used because of their ease of formulation and efficiency from the viewpoint of absorption and bioavailability (See, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich, N.G., and Ansel, H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral oil-based laxatives, fat-soluble vitamins, and high-fat nutrients are generally one of the materials administered orally as o / w emulsions.

[0395] ii. Microemulsion In one embodiment of the present invention, the composition of iRNA and nucleic acid is formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and amphiphilic substances 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, N.Y., volume 1, p. 245). Typically, a microemulsion is a system prepared by first dispersing oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an alcohol of intermediate chain length, to form a transparent system. Thus, a microemulsion has been described as a thermodynamically stable isotropically transparent dispersion of two immiscible liquids stabilized by an interfacial film of surfactant 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 usually prepared through combinations of three to five components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is of the water-in-oil (w / o) or oil-in-water (o / w) type 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 state diagrams has been widely studied and has provided those skilled in the art with comprehensive knowledge regarding the preparation of microemulsions (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, N.Y., volume 1, p. 245; Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs into thermodynamically stable formulations of spontaneously formed small droplets.

[0397] Surfactants used in the preparation of microemulsions include 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), decaglycerol decaoleate (DAO750), either alone or in combination with a co-surfactant, but are not limited thereto. Usually, co-surfactants that are short-chain alcohols such as ethanol, 1-propanol, and 1-butanol help to increase the interfacial fluidity by penetrating into the surfactant coating film, and as a result, create an irregular coating film due to the gaps formed between surfactant molecules. However, microemulsions can be prepared without the use of a co-surfactant, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of a drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. 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 can be used as the oil phase, but are not limited thereto.

[0398] Microemulsions are of particular interest from the perspective 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 such as peptides (see, for example, U.S. Patent No. 6,191,105; U.S. Patent No. 7,063,860; U.S. Patent No. 7,070,802; U.S. Patent No. 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, protection of drugs from enzymatic hydrolysis, expected enhanced drug absorption due to changes in membrane fluidity and permeability induced by surfactants, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Patent No. 6,191,105; U.S. Patent No. 7,063,860; U.S. Patent No. 7,070,802; U.S. Patent No. 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 together at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides or iRNA. Microemulsions have been effective in the transdermal delivery of active ingredients for both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate the increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract, as well as improve the local intracellular uptake of iRNA and nucleic acids.

[0399] The microemulsion of the present invention may also contain additional components 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 microemulsion 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. Microparticles The iRNA agent of the present invention may be incorporated into particles such as, for example, microparticles. Microparticles can be produced by spray drying, but they may also be produced by other methods including freeze drying, evaporation, fluidized bed drying, vacuum drying, or combinations of these techniques.

[0401] iv. Penetration Enhancers In one embodiment, the present invention provides efficient delivery of nucleic acids, particularly iRNA, to animal skin using various penetration enhancers. Most drugs exist in solution in both ionized and non-ionized forms. However, usually only lipid-soluble or lipophilic drugs can easily cross cell membranes. It has been found that non-lipophilic drugs can also cross cell membranes if the membranes to be crossed are treated with penetration enhancers. In addition to assisting 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, namely surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each class of the aforementioned penetration enhancers will be described in more detail below.

[0403] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, lower the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved iRNA absorption through the mucosa. 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, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0404] Examples of various fatty acids and their derivatives that act as penetration enhancers include 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, acylcarnitine, acylcholine, and their C 1~20 alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and di-glycerides (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] Physiological roles of bile include 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), glycocholic acid (sodium glycocholate), glycochenodeoxycholic acid (sodium glycochenodeoxycholate), 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, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 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 remove metal ions from solution by forming a complex therewith, resulting in improved iRNA absorption through the mucosa. With respect to their use as penetration enhancers in the present invention, most DNA nucleases require divalent metal ions for catalysis and are inhibited by chelating agents, so chelating agents have the additional advantage of also acting as deoxyribonuclease inhibitors (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (such as sodium salicylate, 5-methoxysalicylate, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (see, for example, 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] In the usage of this specification, a non-chelating non-surfactant penetration enhancer demonstrates insignificant activity as a chelating agent or as a surfactant, but can still be defined as a compound that enhances the absorption of iRNA through the gastrointestinal mucosa (see, for example, Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of penetration enhancers in this class include unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal 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 uptake of iRNA can also be added to the medicaments and other compositions of the present invention. For example, cationic lipids such as lipofectine (U.S. Patent No. 5,705,188, issued to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97 / 30731, published to Lollo et al.) are also known to enhance the intracellular uptake of dsRNA.Examples of commercially available transfection reagents include, for example, in particular, 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 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), iRNAMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (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® Reagent (Promega; Madison, WI), TransFast™ 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 Examples include D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec (trademark) / LipoGen (trademark) (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 (trademark) transfection Reagent (Genlantis; San Diego, CA, 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 (trademark) (B-Bridge International, Mountain View, CA, USA).

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

[0410] v. carrier Certain compositions of the invention also incorporate a carrier compound during formulation. As used herein, a "carrier compound" or "carrier" is inert (i.e., has no biological activity per se), but can be recognized by a biologically active nucleic acid, e.g., by a biological process that degrades the biologically active nucleic acid or promotes its removal from circulation, thereby reducing the bioavailability of the biologically active nucleic acid. Co-administration of a nucleic acid and a carrier compound, typically in excess of the latter substance, can likely result in a substantial decrease in the amount of nucleic acid recovered in the liver, kidney, or other extravascular storage sites, probably due to competition between the carrier compound and the nucleic acid for normal receptors. For example, the recovery of partially phosphorothioated dsRNA in liver tissue can be decreased when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyanato-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. Excipient In contrast to the carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquids or solids and are selected with the intended mode of administration in mind to provide the desired bulk, viscosity, etc. when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); diluents (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (such as starch, sodium starch glycolate); and wetting agents (such as sodium lauryl sulfate), but are not limited thereto.

[0412] The compositions of the present invention can be formulated using pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with the nucleic acids and are suitable for oral administration. Suitable pharmaceutically acceptable carriers include water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, but are not limited thereto.

[0413] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil bases. The solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not react detrimentally with the nucleic acids and are suitable for oral administration can be used.

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

[0415] vii. Other components The composition of the present invention may further contain other auxiliary agent components found in conventional pharmaceutical compositions at their established usage levels in the art. Thus, for example, the composition can contain additional compatible pharmacologically active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage for...

Claims

1. A salt of a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of transthyretin (TTR) intracellularly, comprising a sense strand and an antisense strand forming a double-stranded region, wherein each of the sense strand and the antisense strand is 21-25 nucleotides in length, the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaga-3' of SEQ ID NO: 10, and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAf aAf ucccasusc-3' of SEQ ID NO: 7, 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 bond, and the sense strand of the double-stranded RNAi agent is bound to a ligand, A salt of the RNAi agent.

2. The salt of the double-stranded RNAi agent according to claim 1, wherein the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker.

3. The ligand is 【Chemical 1】 The salt of the double-stranded RNAi agent according to claim 2.

4. The salt of the double-stranded RNAi agent according to claim 1, wherein the ligand is bound to the 3' end of the sense strand.

5. The RNAi agent is bound to a ligand having the following structure: 【Chemical Formula 2】 and the ligand is as shown in the following schematic diagram [Chemical 3] (wherein X is O or S) The salt of the double-stranded RNAi agent according to claim 4, which is bound to the 3' end of the sense strand as shown.

6. The salt of the double-stranded RNAi agent according to claim 5, wherein X is O.

7. The salt of the double-stranded RNAi agent according to claim 1, wherein the salt is a sodium salt.

8. A salt of a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of transthyretin (TTR) intracellularly, comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand consists of the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaga-3' of SEQ ID NO: 10, and the antisense strand consists of the nucleotide sequence 5'-usCfsuugGfuuAfcaugAf aAf ucccasusc-3' of SEQ ID NO: 7, a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; and the sense strand of the double-stranded RNAi agent is bound to a ligand, a salt of a double-stranded ribonucleic acid (RNAi) agent. **Claim 9** The salt of the double-stranded RNAi agent according to claim 8, wherein the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker. **Claim 10** The ligand is 【Chemical Formula 4】 The salt of the double-stranded RNAi agent according to claim 9. **Claim 11** The salt of the double-stranded RNAi agent according to claim 8, wherein the ligand is bound to the 3'-end of the sense strand. **Claim 12** The RNAi agent is bound to a ligand having the following structure: [Chemical Formula 5] , and the ligand is the following schematic diagram [Chemical Formula 6] (wherein X is O or S) The salt of the double-stranded RNAi agent according to claim 11, wherein the ligand is bound to the 3'-end of the sense strand as shown. **Claim 13** The salt of the double-stranded RNAi agent according to claim 12, wherein X is O. **Claim 14** The salt of the double-stranded RNAi agent according to claim 8, wherein the salt is a sodium salt. **Claim 15** A salt of a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of transthyretin (TTR) intracellularly, comprising a sense strand and an antisense strand forming a double-stranded region, wherein each of the sense strand and the antisense strand is 21-25 nucleotides in length, the sense strand comprises the nucleotide sequence 5'-usggggaUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10, and the antisense strand comprises the nucleotide sequence 5'-usCfsuuGfuuAfcaugAfafaUfccccasusuc-3' of SEQ ID NO: 7, a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate linkage; and the 3'-end of the sense strand of the RNAi agent is bound to a ligand having the following structure: [Chemical Formula 7] , and the ligand is the following schematic diagram 【Chemical 8】 (wherein X is O) The salt of a double-stranded ribonucleic acid (RNAi) agent, wherein the ligand is bound to the 3'-end of the sense strand as shown. **Claim 16** A salt of a double-stranded ribonucleic acid (RNAi) agent that inhibits the expression of transthyretin (TTR) intracellularly, comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand consists of the nucleotide sequence 5'-usgsggaauUfuCfAfUfguaaccaga-3' of SEQ ID NO: 10, and the antisense strand consists of the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaaAfucccasusuc-3' of SEQ ID NO: 7, wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, and U, respectively; s is a phosphorothioate bond; and the 3'-end of the sense strand of the RNAi agent is bound to a ligand having the following structure: [Chemical Formula 9] , and the ligand is the following schematic diagram 【Chemical Formula 10】 (wherein X is O) A salt of a double-stranded ribonucleic acid (RNAi) agent, which is bound to the 3'-end of the sense strand as shown in the above formula.

17. A pharmaceutical composition comprising the salt of the double-stranded RNAi agent according to any one of Claims 1-16.

18. The pharmaceutical composition according to Claim 17, which contains a non-buffer solution.

19. The pharmaceutical composition according to Claim 18, wherein the non-buffer solution is physiological saline or water.

20. The pharmaceutical composition according to Claim 17, which contains a buffer solution.

21. The pharmaceutical composition according to Claim 20, wherein the buffer solution contains acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

22. The pharmaceutical composition according to Claim 20, wherein the buffer solution contains phosphate.

23. A pharmaceutical composition for treating a subject suffering from a TTR-related disease, comprising the salt of the double-stranded RNAi agent according to any one of Claims 1-16, or the pharmaceutical composition according to any one of Claims 17-22.

24. The pharmaceutical composition according to Claim 23, wherein the neuropathy impairment score (NIS) or the revised NIS (mNIS+7) decreases, slows down, or stops in a subject suffering from a TTR-related disease.

25. The pharmaceutical composition according to Claim 23, wherein the 6-minute walk test (6MWT) increases in a subject suffering from a TTR-related disease.

26. The pharmaceutical composition according to Claim 23, wherein the subject is a human.

27. The pharmaceutical composition according to claim 23, wherein the subject has a TTR gene mutation associated with the onset of a TTR-related disease.

28. The pharmaceutical composition according to claim 23, wherein the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia.

29. The pharmaceutical composition according to claim 23, wherein the subject has TTR-related amyloidosis.

30. The pharmaceutical composition according to claim 23, wherein the subject has TTR-related amyloidosis and the pharmaceutical composition treats the polyneuropathy of TTR-related amyloidosis.

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

32. The pharmaceutical composition according to claim 23, wherein the salt of the double-stranded RNAi agent is administered to the subject via subcutaneous administration.

33. The pharmaceutical composition according to claim 32, wherein the subcutaneous administration is self-administration.

34. The pharmaceutical composition according to claim 33, wherein the self-administration is via a prefilled syringe or an autoinjector syringe.

35. The pharmaceutical composition according to claim 23, wherein the salt of the double-stranded RNAi agent is chronically administered to the subject.

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