Method for treating or preventing TTR-related diseases using transthyretin (TTR) iRNA compositions

Abstract

Pharmaceutical compositions for treating human subjects suffering from or at risk of developing a TTR-related disorder are provided. [Solution] A pharmaceutical composition comprising a double-stranded RNAi agent administered at a fixed dose of approximately 50 mg, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 435,127, filed on 16 December 2016, the entirety of which is incorporated herein by reference.

[0002] This application relates to International Application PCT / US2016 / 044359 filed on 28 November 2016, U.S. Provisional Patent Application No. 62 / 199,563 filed on 31 July 2015, and U.S. Provisional Patent Application No. 62 / 287,518 filed on 27 January 2016. The entire contents of each of the aforementioned applications are incorporated herein by reference.

[0003] This application also relates to U.S. Provisional Patent Application No. 61 / 881,257, filed on 23 September 2013, and to International Application PCT / US2014 / 056923, filed on 23 September 2014 (the entire contents of each of these applications are incorporated herein by reference). Furthermore, this application relates to U.S. Provisional Patent Application No. 61 / 561,710 filed on 18 November 2011, International Application PCT / US2012 / 065601 filed on 16 November 2012, U.S. Provisional Patent Application No. 61 / 615,618 filed on 26 March 2012, U.S. Provisional Patent Application No. 61 / 680,098 filed on 6 August 2012, U.S. Patent Application No. 14 / 358,972 filed on 16 May 2014, and International Application PCT / US2012 / 065691 filed on 16 November 2012 (the entire contents of each of these are incorporated herein by reference).

[0004] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on December 7, 2017, is named 121301-07020_SL.txt and has a size of 9,480 bytes. [Background technology]

[0005] Transthyretin (TTR), also known as prealbumin, is found in serum and cerebrospinal fluid (CSF). TTR transports retinol-binding protein (RBP) and thyroxine (T4), and further acts as a carrier of retinol (vitamin A) through its association with RBP in the blood and CSF. Transthyretin is named after its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apoAI (major HDL apolipoprotein), amyloid-beta peptide, and neuropeptide Y. See Non-Patent Literature 1.

[0006] TTR is a tetramer of four identical 127-amino acid subunits (monomers) abundantly present within the β-sheet structure. Each monomer has two 4-chain β-sheets and an elongated spherical shape. Antiparallel β-sheet interactions link monomers to dimers. Short loops from each monomer form the main dimer-dimer interactions. These two loop pairs separate the opposing convex β-sheets of the dimer, forming internal channels.

[0007] The liver is the primary site of TTR expression. Other prominent sites of expression include the choroid plexus, retina (particularly retinal pigment epithelium), and pancreas.

[0008] Transthyretin is one of at least 27 different types of proteins that are precursor proteins in amyloid fibril formation. See Non-Patent Literature 2. Extracellular deposition of amyloid fibrils in organs and tissues is characteristic of amyloidosis. Amyloid fibrils consist of misfolded protein aggregates, which may result from either excessive production 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 Literature 3.

[0009] 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 begin with "A" for amyloid, followed by an abbreviation of the precursor protein, such as ATTR, which corresponds to amyloidogenic transthyretin (ibid.).

[0010] A great many TTR-related diseases exist, the majority of which are amyloid diseases. Normal-sequence TTR is associated with cardiac amyloidosis in older adults and is called senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA is often accompanied by microscopic deposition in many other organs. TTR amyloidosis manifests in various forms. When the peripheral nervous system is more prominently affected, the disease is called familial amyloid polyneuropathy (FAP). When the heart is primarily involved but the nervous system is not, the disease is called familial amyloid cardiomyopathy (FAC). A third major type of TTR amyloidosis is leptomeningeal amyloidosis (also known as leptomeningeal or meningeal vascular amyloidosis), central nervous system (CNS) amyloidosis, or amyloidosis type VII. Mutations in TTR can also cause amyloid vitreous opacity, carpal tunnel syndrome, and normal thyroid hyperthyroxinemia (a non-amyloid disorder thought to be secondary to increased association between thyroxine and TTR due to mutant TTR molecules with increased affinity for thyroxine). See, for example, Non-Patent Document 4.

[0011] Abnormal amyloid-forming proteins can be inherited or acquired through somatic mutations (Non-Patent Literature 2). Transthyretin-associated ATTR is the most common form of hereditary systemic amyloidosis (Non-Patent Literature 5). TTR mutations accelerate 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, but some mutations are associated with cardiomyopathy or vitreous opacity (ibid.).

[0012] 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 of age (Non-Patent Document 7). [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Liz, MAet al. (2010) IUBMB Life,62(6):429-435 [Non-Patent Document 2] Guan,J.et al.(Nov.4,2011)Current perspectives on cardiac amyloidosis,Am J Physiol Heart Circ Physiol,doi:10.1152 / ajpheart.00815.2011 [Non-Patent Document 3] Saraiva MJM(2002)Expert Reviews in Molecular Medicine,4(12):1-11 [Non-Patent Document 4] Moses et al.(1982)J.Clin.Invest.,86,2025-2033 [Non-Patent Document 5] Lobato, L. (2003) J. Nephrol., 16:438-442 [Non-Patent Document 6] Jacobson, DRet al. (1997) N. Engl. J. Med. 336(7):466-73 [Non-Patent Document 7] Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. USA87(7):2843-5 [Overview of the project] [Problems that the invention aims to solve]

[0014] Therefore, there is a need for effective treatments for TTR-related diseases in this technological field. [Means for solving the problem]

[0015] The present invention provides a method for inhibiting TTR expression, and a method for treating or preventing transthyretin (TTR)-related diseases in human subjects using RNAi agents that target the TTR gene, such as double-stranded RNAi agents. The present invention is at least in part based on the discovery shown herein that RNAi agents comprising substantially all nucleotides on the sense strand and substantially all nucleotides on the antisense strand are modified nucleotides, and that include 8 or fewer 2'-fluoro modifications on the sense strand, 6 or fewer 2'-fluoro modifications on the antisense strand, two phosphorothioate bonds at the 5' end of the sense strand, two phosphorothioate bonds at the 5' end of the antisense strand, and a ligand, such as GalNAc3 ligand, are effective in terminating the activity of the TTR gene. These agents exhibit surprisingly enhanced TTR gene silencing activity. Unless intended to be theoretically limiting, the aforementioned modifications and specific target site combinations or partial combinations in these RNAi agents are thought to confer improved efficacy, stability, potency, and durability to the RNAi agents of the present invention.

[0016] Accordingly, in one embodiment, the present invention provides a method for treating a human subject who has or is at risk of developing a TTR-related disease. This method comprises administering a double-stranded RNAi agent to a human subject in a constant dose of about 25 mg to about 50 mg (e.g., about 25, 30, 35, 40, 45, or about 50 mg), wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-u The compound comprises sCfsuugGfuuAfcaugAfaAfucccasusc-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'-fluoroA, C, G, or U; and s is a phosphorothioate bond), thereby treating human subjects who have or are at risk of developing TTR-related disease.

[0017] In another aspect, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease. The method involves administering a double-stranded RNAi agent to a human subject at a constant dose of approximately 25 mg to approximately 50 mg, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7) (wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate bond), thereby improving at least one indicator of neurological impairment or quality of life in a human subject.

[0018] In one embodiment, the index is a neuropathy index, such as the Neuropathy Indication (NIS) score or the Revised NIS (mNIS+7) score. In another embodiment, the index is a quality of life index selected from the group consisting of, for example, the SF-36® health survey score, the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, the NIS-W score, the Rasch-built Overall Disability Scale (R-ODS) score, the Composite Autonomic Symptoms Score (COMPASS-31), the Median Body Mass Index (mBMI) score, the 6-Minute Walk Test (6MWT) score, and the 10-meter Walk Test score.

[0019] In another embodiment, the present invention provides a method for reducing, delaying, or halting the neurological impairment score (NIS) or revised NIS (mNIS+7) in human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject in a constant dose of about 25 mg to about 50 mg (e.g., about 25, 30, 35, 40, 45, or about 50 mg), wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuu The compound gGfuuAfcaugAfaAfucccasusc-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'-fluoroA, C, G, or U; and s is a phosphorothioate bond), thereby reducing, delaying, or halting the neurological impairment score (NIS) or revised NIS (mNIS+7) in human subjects.

[0020] In another aspect, the present invention provides a method for increasing the 6-minute walk test (6MWT) in human subjects who have or are at risk of developing TTR-related disease. The method involves administering a double-stranded RNAi agent to human subjects at a constant dose of approximately 25 mg to approximately 50 mg (e.g., approximately 25, 30, 35, 40, 45, or approximately 50 mg), wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, where the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7) (wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate bond), thereby increasing the 6-minute walk test (6MWT) in human subjects.

[0021] In one embodiment, the human subject is a human subject suffering from a TTR-related disease. In another embodiment, the human subject is a human subject at risk of developing a TTR-related disease. In yet another embodiment, the human subject has a TTR gene mutation associated with the development of a TTR-related disease. In one embodiment, the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), pia mater / central nervous system (CNS) amyloidosis, and hyperthyroxinemia. In yet another embodiment, the human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the method reduces amyloid TTR deposition in the human subject. ATTR may be hereditary ATTR (h-ATTR) or non-hereditary ATTR (wt ATTR).

[0022] Double-stranded RNAi agents may be administered to human subjects by means of administration selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic vessel, cerebrospinal fluid, and any combination thereof.

[0023] In one embodiment, the double-stranded RNAi agent is administered to human subjects via subcutaneous, intramuscular, or intravenous administration. In another embodiment, the double-stranded RNAi agent is administered to human subjects via subcutaneous administration. In one embodiment, subcutaneous administration is self-administered. In one embodiment, self-administered is via a pre-filled syringe or an autoinjector syringe.

[0024] The method of the present invention may further include evaluating the level of TTR mRNA expression or TTR protein expression in a sample derived from a human subject.

[0025] Double-stranded RNAi agents may be administered to human subjects every 3 months, 4 months, 5 months, or 6 months. In one embodiment, a fixed dose of the double-stranded RNAi agent is administered to human subjects once every 3 months. In another embodiment, a fixed dose of the double-stranded RNAi agent is administered to human subjects once every 6 months.

[0026] In one embodiment, the double-stranded RNAi agent is administered chronically to human subjects.

[0027] In one embodiment, the double-stranded RNAi agent is administered to human subjects at a constant dose of approximately 25 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects at a constant dose of approximately 50 mg.

[0028] In one embodiment, the method of the present invention may further include providing additional therapeutic measures, for example, for orthotopic liver transplantation, pacemaker implantation, and heart transplantation, and / or administering additional therapeutic agents useful for treating TTR-related diseases, for example, TTR tetramer stabilizers, for example, tafamidis, and / or nonsteroidal anti-inflammatory drugs (NSAIDs), for example, diflunisal, to human subjects.

[0029] In one embodiment, the sense strand of a double-stranded RNAi agent is conjugated to at least one ligand.

[0030] In one embodiment, the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker.

[0031] In one embodiment, the ligand is [ka] That is the case.

[0032] In one embodiment, the ligand is bound to the 3' end of the sense chain.

[0033] In one embodiment, the RNAi agent is conjugated to a ligand as shown in the schematic diagram below. [ka] (In the formula, X is either O or S)

[0034] In one embodiment, the sense strand of the RNAi agent comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (SEQ ID NO: 15), and the antisense strand of the RNAi agent comprises 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'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol).

[0035] In one embodiment, the present invention provides a method for treating a human subject who has or is at risk of developing a TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 25 mg about once every three months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (Sequence ID 17), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (Sequence ID 17). Formula 7) comprises (wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby treating human subjects who have or are at risk of developing TTR-related disease.

[0036] In another embodiment, the present invention provides a method for treating human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 25 mg about once every 6 months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (Sequence ID 15), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (Sequence ID 15). Formula 7) comprises (wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby treating human subjects who have or are at risk of developing TTR-related disease.

[0037] In one embodiment, the present invention provides a method for treating a human subject who has or is at risk of developing a TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 50 mg about once every three months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (Sequence ID 15), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (Sequence ID 15). Formula 7) comprises (wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby treating human subjects who have or are at risk of developing TTR-related disease.

[0038] In another embodiment, the present invention provides a method for treating human subjects who have or are at risk of developing TTR-related diseases. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 50 mg about once every 6 months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (Sequence ID 15), and the antisense strand comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (Sequence ID 15). Formula 7) comprises (wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby treating human subjects who have or are at risk of developing TTR-related disease.

[0039] In one embodiment, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 25 mg about once every three months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (SEQ ID NO: 15), and the antisense strand comprises 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'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease.

[0040] In another embodiment, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 25 mg about once every 6 months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (SEQ ID NO: 15), and the antisense strand comprises 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'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease.

[0041] In one embodiment, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 50 mg about once every three months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (SEQ ID NO: 15), and the antisense strand comprises 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'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease.

[0042] In another embodiment, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a double-stranded RNAi agent to a human subject at a constant dose of about 50 mg about once every six months, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, wherein the sense strand comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (SEQ ID NO: 15), and the antisense strand comprises 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'-fluoroA, C, G, or U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidedecanoyl)]-4-hydroxyprolinol), thereby improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease.

[0043] In one embodiment, the index is a neuropathy index, such as the Neuropathy Indication (NIS) score or the Revised NIS (mNIS+7) score. In another embodiment, the index is a quality of life index selected from a group consisting of, for example, the SF-36® Health Survey score, the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, the NIS-W score, the Rasch-built Overall Disability Scale (R-ODS) score, the Composite Autonomic Symptoms Score (COMPASS-31), the Median Body Mass Index (mBMI) score, the 6-Minute Walk Test (6MWT) score, and the 10-meter Walk Test score.

[0044] The present invention further provides a kit for carrying out any of the methods of the present invention. The kit may include a double-stranded RNAi agent and a label containing instructions for use.

[0045] The present invention is further illustrated by the following detailed description and drawings. [Brief explanation of the drawing]

[0046] [Figure 1] Figure 1 is a graph showing the suppression of TTR protein in healthy human volunteers who received AD-65492 as a single dose of 5 mg, 25 mg, 50 mg, 100 mg, 200 mg, or 300 mg subcutaneously. The graph shows the mean [+ / - standard error of mean] TTR relative to baseline over time for the cohort. [Modes for carrying out the invention]

[0047] The present invention provides a method for inhibiting TTR expression using an RNAi agent that targets the TTR gene, such as a double-stranded RNAi agent, and a method for treating or preventing transthyretin (TTR)-related diseases in human subjects. The present invention is at least in part based on the finding shown herein that RNAi agents comprising substantially all nucleotides on the sense strand and substantially all nucleotides on the antisense strand are modified nucleotides, and that the sense strand 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, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications), the antisense strand has 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), two phosphorothioate bonds at the 5' end of the sense strand, two phosphorothioate bonds at the 5' end of the antisense strand, and a ligand, e.g., GalNAc3 ligand, are effective in selectively terminating the activity of the TTR gene. These agents exhibit surprisingly enhanced TTR gene silencing activity. Unless intended to impose theoretical limitations, the aforementioned modifications and specific target site combinations or partial combinations in these RNAi agents are expected to confer improved efficacy, stability, potency, and durability to the RNAi agents of the present invention.

[0048] The following detailed description discloses methods for preparing and utilizing compositions containing iRNAs that selectively inhibit TTR gene expression, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from inhibition and / or reduction of TTR gene expression.

[0049] I. Definition To make the present invention easier to understand, certain terms are defined first. In addition, whenever parameter values ​​or ranges of values ​​are enumerated, it should be noted that intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of the present invention.

[0050] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical purposes of the article. For example, "an element" means one factor or two or more factors, such as multiple factors.

[0051] The term "including" is used herein to mean "including, but not limited to," and is used without distinction from that term.

[0052] In this specification, the term "or" is used to mean, and is used without distinction from, the term "and / or" unless the context clearly indicates otherwise.

[0053] The term “approximately” is used herein to mean a typical range acceptable in the art. For example, “approximately” may be understood as within approximately two standard deviations from the mean. In certain embodiments, “approximately” means +10%. In certain embodiments, “approximately” means +5%. When “approximately” precedes a set of numbers or a range, it is understood that “approximately” may modify each of the numbers or the range.

[0054] 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 bloodstream, 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, which become extracellular deposits, causing amyloidosis. For a review, see, for example, Saraiva MJM (2002) Expert Reviews in Molecular Medicine, 4(12):1-11. Molecular cloning and nucleotide sequences of rat transthyretin, as well as the distribution of mRNA expression, are described by Dickson, P. Wet. 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. Sequences of human TTR mRNA transcripts can be found in the National Center for Biotechnology Information (NCBI) RefSeq access number NM_000371 (e.g., SEQ ID NOs. 1 and 5). Sequences of mouse TTR mRNA can be found in RefSeq access number NM_013697.2, and sequences of rat TTR mRNA can be found in RefSeq access number NM_012681.1. Further examples of TTR mRNA sequences are readily available in publicly available databases, such as Genbank, UniProt, and OMIM.

[0055] When used herein, “TTR-related disease” is intended to include any disease associated with the TTR gene or protein. Such diseases may be caused, for example, by excessive production of the TTR protein, by mutations in the TTR gene, 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 transthyretin-mediated amyloidosis (ATTR amyloidosis), in which TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposition, such as hereditary ATTR (h-ATTR) or non-hereditary ATTR (wt ATTR). TTR-related disorders include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), pia mater / central nervous system (CNS) amyloidosis, amyloid vitreous opacity, carpal tunnel syndrome, and hyperthyroxinemia. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, hypoesthesia in the distal extremities), autonomic neuropathy (e.g., gastrointestinal disorders, e.g., gastric ulcers, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial neuropathy, and corneal lattice dystrophy.

[0056] In the use of this specification, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TTR gene, including mRNA, which is the RNA processing product of the primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage, either in or near the portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TTR gene. In one embodiment, the target sequence is located within the protein-coding region of the TTR gene. In another embodiment, the target sequence is located within the 3'UTR of the TTR gene.

[0057] The target sequence may be approximately 9 to 36 nucleotides long, for example, approximately 15 to 30 nucleotides long. For example, the target sequences may be 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-2 6, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, etc., which may be about 15-30 nucleotides long. In some embodiments, the target sequence is about 19-30 nucleotides long. In other embodiments, the target sequence is about 19-25 nucleotides long. In yet another embodiment, the target sequence is about 19-23 nucleotides long. In some embodiments, the target sequence is about 21-23 nucleotides long. Intermediate ranges and lengths between those listed above are also considered to be part of the present invention.

[0058] In some embodiments of the present invention, the target sequence of the TTR gene includes 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).

[0059] In the use of this specification, the term “sequence-containing chain” refers to an oligonucleotide containing a nucleotide chain described by the sequence referred to, using standard nucleotide nomenclature.

[0060] "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 substituted portions, as will be further detailed below (see, for example, Table 2). Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be replaced with other portions without substantially altering the base-pairing properties of oligonucleotides containing such substituted portions. As an example not intended to be limiting, a nucleotide containing inosine as a base can base-pair with adenine, cytosine, or uracil-containing nucleotides. Thus, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the dsRNAs discussed in this invention with, for example, nucleotides containing inosine. In another embodiment, adenine and cytosine may be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form GU fluctuation base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods discussed in this invention.

[0061] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interfering agent” are used synonymously herein and refer to agents containing RNA as defined herein that mediate 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 those in mammalian subjects.

[0062] In one embodiment, the RNAi agent of the present invention is a single-stranded RNA that interacts with a target RNA sequence, such as a TTR target mRNA sequence, to induce cleavage of the target RNA. Although we do not want to be constrained by theory, it is thought that a long double-stranded RNA introduced into a cell is degraded by a type III endonuclease 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 a 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Next, these siRNAs are incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing a 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 the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one embodiment, the present invention relates to single-stranded siRNA (ssRNA) (antisense strand of siRNA double strand) that is generated intracellularly and promotes the formation of a RISC complex to silence a target gene, namely the TTR gene. Accordingly, the term "siRNA" is also used herein to refer to RNAi as described above.

[0063] In another embodiment, the RNAi agent may be a single-stranded RNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaut 2 and then cleaves the target mRNA. Single-stranded siRNAs are typically 15 to 30 nucleotides and are chemically modified. Designs and tests of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, their entire contents of which are incorporated herein by reference. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein or chemically modified in the manner described in Lima et al., (2012) Cell 150:883-894.

[0064] In another embodiment, the “iRNA” used in the compositions, uses, and methods of the present invention is double-stranded RNA and is referred herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a double-stranded ribonucleic acid molecule complex having a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having “sense” and “antisense” orientations with respect to the target RNA, i.e., the TTR gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0065] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, “RNAi agent” may contain chemically modified ribonucleotides; and RNAi agent may contain substantial modifications to multiple nucleotides.

[0066] As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified nucleotide-nucleotide bond, and / or a modified nucleic acid base. Therefore, the term “modified nucleotide” encompasses substitution, addition, or removal of, for example, functional groups or atoms to the nucleoside bond, sugar moiety, or nucleic acid base. Modifications suitable for use in the active substance of the present invention include all types of modifications disclosed herein or known in the art. Any such modification, when used in an siRNA-type molecule for the purposes of this application and claims, is encompassed by “RNAi agent.”

[0067] The double-stranded region may be of any length that enables the specific degradation of the desired target RNA via the RISC pathway, and may be in the range of approximately 9 to 36 base pairs in length, for example, approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example, approximately 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, These include base pair lengths of 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0068] The two strands forming the double-stranded structure may be different parts of a larger RNA molecule, or they may be different RNA molecules. When the two strands are parts of one larger molecule, and thus the 3' end of one strand forming the double-stranded structure is joined to the 5' end of the other strand by an uninterrupted nucleotide strand, the joined RNA strand is called a “hairpin loop”. A hairpin loop may contain at least one unpaired nucleotide; in some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, and at least 23 or more unpaired nucleotides. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 nucleotides.

[0069] If two substantially complementary strands of dsRNA are composed of another RNA molecule, these molecules may, but do not necessarily, be covalently linked. If the two strands are covalently linked between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand by means other than an uninterrupted nucleotide chain, the linking structure is called a "linker." 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 dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs.

[0070] 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, with each strand being 24-30 nucleotides long. Although we do not wish to be constrained by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer's ribonuclease III-like enzyme processes the dsRNA to yield a short interfering RNA of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing a complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment of the RNAi agent, at least one strand 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, e.g., nucleotides 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15. In yet another embodiment, 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, for example, nucleotides 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent contain an overhang of at least one nucleotide.

[0071] In one embodiment, the RNAi agent of the present invention is a dsRNA that interacts with a TTR RNA sequence to induce cleavage of a target RNA, with each strand containing 19–23 nucleotides. Although we do not wish to be constrained by theory, the long double-stranded RNA introduced into the cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer's ribonuclease III-like enzyme processes the dsRNA to yield a short interfering RNA of 19–23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing a complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with the TTR RNA sequence to induce cleavage of the target RNA.

[0072] 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 dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of dsRNA extends over the 5' end of the other strand, or vice versa. A 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, or at least five or more nucleotides. A nucleotide overhang may contain, or consist of, nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, the 3' end, or both ends of either the antisense or sense strand of the dsRNA. In one embodiment of 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, for example, nucleotides 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15. In yet another embodiment, at least one strand of the RNAi agent contains a 5' overhang of at least one nucleotide. In a particular embodiment, at least one strand contains a 5' overhang of at least two nucleotides, for example, nucleotides 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent contain an overhang of at least one nucleotide.

[0073] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, such as 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging, for example, at the 3' and / or 5' ends. In another embodiment, one or more nucleotides in the overhang are substituted with thiophosphate nucleosides.

[0074] In certain embodiments, the overhang on the sense strand or the antisense strand, or both, may include nucleotide lengths longer than 10, e.g., 1–30 nucleotides, 2–30 nucleotides, 10–30 nucleotides, or 10–15 nucleotides. In certain embodiments, the extended overhang is located on the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located on the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded antisense strand. In certain embodiments, one or more nucleotides in the overhang are replaced with nucleoside thiophosphates. 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.

[0075] "Blunt-terminated" or "blunt-ended" means that a double-stranded RNAi agent does not have an unpaired nucleotide at its end, i.e., it does not have a nucleotide overhang. A "blunt-terminated" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., 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 active substance having one overhang and one blunt end) or RNAi agents having nucleotide overhangs at both ends.

[0076] The terms “antisense strand” or “guide strand” refer to a strand of iRNA, e.g., dsRNA, that contains a region substantially complementary to the target sequence, e.g., TTR mRNA. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to the sequence, e.g., the target sequence, e.g., TTR nucleotide sequence, as defined herein. If the complementarity of the complementary region to the target sequence is not perfect, the mismatch may be located within the molecule or in the terminal region. Generally, the most acceptable mismatches are located in the terminal region, e.g., in the 5th, 4th, 3rd, 2nd, or one nucleotide at the 5' and / or 3' ends of the iRNA. In one embodiment, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the antisense strand. In another embodiment, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is located, e.g., in the 5th, 4th, 3rd, 2nd, or the first nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is located, e.g., in the 3' terminal nucleotide of the iRNA.

[0077] The terms “sense strand” or “passenger strand,” as used herein, refer to a strand of iRNA containing a region substantially complementary to the antisense strand region as defined herein.

[0078] As used herein, the term “cleavage region” refers to a region directly adjacent to a cleavage site. A cleavage site is a site on the target where cleavage occurs. In some embodiments, the cleavage region includes three bases directly adjacent to either end of the cleavage site. In some embodiments, the cleavage region includes two bases directly adjacent to either end of the cleavage site. In some embodiments, the cleavage site is particularly located at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0079] In the use of this specification, unless otherwise specified, the term “complementary” refers to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under specific conditions to form a double-stranded structure, as will be understood by those skilled in the art when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically reasonable conditions that may be encountered in living organisms, may also be applicable. Those skilled in the art can determine the optimal set of conditions for the complementarity test of the two sequences, depending on the end use of the hybridized nucleotides.

[0080] For example, complementary sequences within iRNAs, such as those within dsRNAs, as described herein, include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence, spanning the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred to herein as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, generally 5, 4, 3, or 2 or fewer, while retaining the ability to hybridize into double-stranded hybridization of up to 30 base pairs under conditions most appropriate for their end use, such as inhibition of gene expression via the RISC pathway. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, where the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, is still referred to as “perfectly complementary” for the purposes described herein.

[0081] "Complementary" sequences, as used herein, also include, or may be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, insofar as the above requirements regarding their hybridizing ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairs or Hoogsteen-type base pairs.

[0082] In this specification, the terms “complementary,” “fully complementary,” and “substantially complementary” may be used in relation to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand and target sequence of an iRNA agent, as will be understood from the context in which they are used.

[0083] As used herein, a polynucleotide "substantially complementary to at least a portion" of messenger RNA (mRNA) means a polynucleotide substantially complementary to the contiguous 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 TTR mRNA if its sequence is substantially complementary to the uninterrupted portion of the mRNA encoding the TTR gene.

[0084] Therefore, 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).

[0085] In other embodiments, the antisense polynucleotides disclosed herein include adjacent nucleotide sequences that 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 any one fragment of SEQ ID NOs: 1, 2, and 5, 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.

[0086] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide (and the antisense polynucleotide is further complementary to a target TTR sequence), wherein the sense strand polynucleotide comprises adjacent nucleotide sequences that are at least about 80% complementary over their entire length to a corresponding region of any one nucleotide sequence in Table 1, 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.

[0087] In another embodiment, the RNAi agent of the present invention comprises an antisense strand substantially complementary to a target TTR sequence and comprises adjacent nucleotide sequences that are at least about 80% complementary over their entire length to a corresponding region of any one nucleotide sequence in Table 1, 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.

[0088] In some embodiments, the majority of the nucleotides in each chain are generally ribonucleotides, but as described in detail herein, one or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, “iRNA” can refer to chemically modified ribonucleotides. 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 encompassed by “iRNA” in its use in an iRNA molecule.

[0089] In one aspect of the present invention, the active substance used in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to the sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base-pairing with mRNA and physically interfering with the translation mechanism; 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 long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may contain a sequence of at least about 15, 16, 17, 18, 19, 20, or more adjacent nucleotides from any one of the antisense sequences described herein.

[0090] II. Methods for treating or preventing TTR-related diseases The present invention provides a method for treating or preventing TTR-related diseases in human subjects, such as transthyretin-mediated amyloidosis (ATTR amyloidosis), such as hereditary ATTR (h-ATTR) or non-hereditary ATTR (wt ATTR). The method comprises administering the RNAi agent of the present invention to the subject in a therapeutically effective or prophylactically effective dose.

[0091] In one embodiment, the present invention provides a method for treating a human subject who has or is at risk of developing a TTR-related disease. The method comprises administering a constant dose of the double-stranded RNAi agent of the present invention to a human subject in a dose of about 25 mg to about 50 mg (e.g., about 25, 30, 35, 40, 45, or about 50 mg).

[0092] In another aspect, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a constant dose of the double-stranded RNAi agent of the present invention to a human subject in a dose of about 25 mg to about 50 mg (e.g., about 25, 30, 35, 40, 45, or about 50 mg).

[0093] In another aspect, the present invention provides a method for reducing, delaying, or halting the neurological impairment score (NIS) or revised NIS (mNIS+7) in human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a constant dose of the double-stranded RNAi agent of the present invention to a human subject in a range of about 25 mg to about 50 mg (e.g., about 25, 30, 35, 40, 45, or about 50 mg).

[0094] In another aspect, the present invention provides a method for increasing the performance of a 6-minute walk test (6MWT) in human subjects who have or are at risk of developing a TTR-related disease. The method comprises administering a constant dose of the double-stranded RNAi agent of the present invention to a human subject of about 25 mg to about 50 mg (e.g., about 25, 30, 35, 40, 45, or about 50 mg).

[0095] In one embodiment, the subjects are: humans being treated or evaluated for a disease, disorder or condition that would benefit from a reduction in TTR gene expression as described herein; humans at risk for a disease, disorder or condition that would benefit from a reduction in TTR gene expression; humans having a disease, disorder or condition that would benefit from a reduction in TTR gene expression; and / or humans being treated for a disease, disorder or condition that would benefit from a reduction in TTR gene expression.

[0096] In some embodiments, the human subjects have a TTR-related disease. In other embodiments, the subjects are at risk of developing a TTR-related disease, for example, subjects with a TTR gene mutation associated with the development of a TTR-related disease (e.g., prior to the onset of signs or symptoms suggestive of TTR amyloidosis), subjects with a family history of a TTR-related disease (e.g., prior to the onset of signs or symptoms suggestive of TTR amyloidosis), or subjects with signs or symptoms suggestive of TTR amyloidosis.

[0097] When used herein, "TTR-related disease" includes any disease caused by or associated with the formation of amyloid deposits, such that the fibrillary precursor consists of mutant or wild-type TTR proteins. Mutant and wild-type TTRs give rise to various forms of amyloid deposition (amyloidosis). Amyloidosis includes the formation and aggregation of misfolded proteins, resulting in extracellular deposition that impairs organ function. Clinical syndromes with TTR aggregation include, for example, senile systemic amyloidosis (SSA); systemic familial amyloidosis; familial polyamyloid neuropathy (FAP); familial amyloid cardiomyopathy (FAC); and leptomania, central nervous system (CNS) amyloidosis, or amyloidosis type VII, also known as leptomania or meningocerebral vascular amyloidosis.

[0098] In one embodiment, the RNAi agent of the present invention is administered to a subject suffering from familial amyloid cardiomyopathy (FAC). In another embodiment, the RNAi agent of the present invention is administered to a subject suffering from FAC with a mixed phenotype, i.e., a subject with both cardiac and neurological disorders. In yet another embodiment, the RNAi agent of the present invention is administered to a subject suffering from FAP with a mixed phenotype, i.e., a subject with both neurological and cardiac disorders. In one embodiment, the RNAi agent of the present invention is administered to a subject suffering from FAP who is being treated with orthotopic liver transplantation (OLT). In another embodiment, the RNAi agent of the present invention is administered to a subject suffering from senile systemic amyloidosis (SSA). In another embodiment of the method of the present invention, the RNAi agent of the present invention is administered to a subject suffering from familial amyloid cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Normal TTR sequences cause cardiac amyloidosis in the elderly, known as senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA is often accompanied by microscopic deposition in many other organs. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of clinically significant TTR amyloidosis (also known as ATTR (amyloidosis-transthyretin type)). More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.

[0099] In some embodiments of the method of the present invention, the RNAi agent of the present invention is administered to subjects suffering from transthyretin (TTR)-associated familial amyloid polyneuropathy (FAP). Such subjects may also suffer from ocular signs, such as vitreous opacity and glaucoma. It is known to those skilled in the art that amyloidogenic transthyretin (ATTR), synthesized by retinal pigment epithelium (RPE), plays a crucial role in the progression of ocular amyloidosis. Previous studies have shown that panretinal laser photocoagulation reduces RPE cells and inhibits the progression of amyloid deposition in the vitreous humor, suggesting that effective suppression of ATTR expression in RPE may be a novel therapeutic approach for ocular amyloidosis (see, e.g., Kawaji, T., et al., Ophthalmology. (2010) 117:552-555). The method of the present invention is useful for the treatment of ocular signs of TTR-associated FAP, such as ocular amyloidosis. RNAi agents can be delivered in a manner suitable for targeting specific tissues, such as the eye. Ocular delivery methods include retrobulbar, subcutaneous, subconjunctival, sub-Tenon's capsule, anterior chamber, or intravitreal injection (or internal injection or infusion). Specific formulations for ocular delivery include eye drops or ointments.

[0100] Another TTR-related disorder is hyperthyroxinemia, also known as "abnormal transthyretinemia" or "abnormal prealbuminemia." This type of hyperthyroxinemia may be secondary to increased association between thyroxine and TTR due to mutant TTR molecules with increased affinity for thyroxine. See, for example, Moses et al. (1982) J.Clin.Invest., 86, 2025-2033.

[0101] The RNAi agents of the present invention may be administered to subjects by any mode of administration known in the art, including, but not limited to, subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic vessel, cerebrospinal fluid, and any combination thereof.

[0102] In a preferred embodiment, the active ingredient is administered subcutaneously to the subject.

[0103] In some embodiments, the RNAi agent is administered to the subject in a single dose via subcutaneous injection, for example, via abdominal, thigh, or upper arm injection. In other embodiments, the RNAi agent is administered to the subject in divided doses via subcutaneous injection. In one embodiment, the RNAi agent is administered to the subject in divided doses via subcutaneous injection to two different anatomical sites in the subject. For example, the subject may receive a divided dose of approximately 25 mg (for example, about half of a 50 mg dose) subcutaneously in the right arm and approximately 25 mg subcutaneously in the left arm. In some embodiments of the present invention, subcutaneous administration is self-administered, for example, via a pre-filled syringe or an auto-injector syringe. In some embodiments, the dose of the RNAi agent for subcutaneous administration is contained in a volume of, for example, 1 ml or less of a pharmaceutically acceptable carrier.

[0104] In some embodiments, administration is via accumulation injection. Accumulation injection may release the RNAi agent consistently over a long period. Therefore, accumulation injection may reduce the frequency of administration required to obtain desired effects, such as desired TTR inhibition or therapeutic or prophylactic effects. Accumulation injection may also provide more consistent serum concentrations. Accumulation injections may be administered subcutaneously or intramuscularly. In preferred embodiments, the accumulation injection is subcutaneous.

[0105] In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneous osmotic pump. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusion. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver.

[0106] In embodiments in which the RNAi agent is administered via a subcutaneous infusion pump, the RNAi agent may be administered to the subject in a single dose over a period of approximately 45 minutes to approximately 5 minutes, for example, approximately 45 minutes, approximately 40 minutes, approximately 35 minutes, approximately 30 minutes, approximately 25 minutes, approximately 20 minutes, approximately 15 minutes, approximately 10 minutes, or approximately 5 minutes.

[0107] Other modes of administration include epidural, intracerebral, intraventricular, nasal, intra-arterial, intracardiac, intraosseous, intrathecal, intravitreous, and pulmonary. The mode of administration may be selected based on whether local or systemic treatment is desired and on the area to be treated. The route and site of administration may be selected to enhance targeting.

[0108] In some embodiments, the RNAi agent is administered to the subject in an amount effective to inhibit TTR expression in cells within the subject. The amount effective to inhibit TTR expression in cells within the subject may be evaluated using methods discussed below, such as methods including evaluation of TTR mRNA, TTR protein, or related variables, such as inhibition of amyloid deposition.

[0109] In some embodiments, the RNAi agent is administered to the subject in a therapeutic or prophylactic effective dose.

[0110] When used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to result in treatment of the disease (for example, by reducing, relieving, or maintaining the symptoms of the pre-existing disease or one or more symptoms of the disease) when administered to a patient for the treatment of a TTR-related disease. “Therapeutic dose” may vary depending on the RNAi agent, the manner in which the agent is administered, the disease and its severity and history, age, weight, family history, genetic structure, stage of the pathological process mediated by TTR expression, the type of preceding or accompanying treatment (if any), and other individual characteristics of the patient to be treated.

[0111] When used herein, “prophylactic effective dose” is intended to include an amount of RNAi agent sufficient to prevent or induce remission of the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or exhibited symptoms of TTR-related disease but is potentially predisposed to the disease. Symptoms that may be induced into remission include sensory neuropathy (e.g., paresthesia, hypoesthesia in the distal extremities), autonomic neuropathy (e.g., gastrointestinal dysfunction, e.g., gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction, and lattice corneal dystrophy. Inducing remission of the disease includes delaying the course of the disease or reducing the severity of later-developing disease. The “effective prophylactic dose” may vary depending on the RNAi agent, the method of administration of the agent, the degree of disease risk, and the patient’s medical history, age, weight, family history, genetic makeup, type of prior or accompanying treatment (if any), and other individual characteristics of the patient being treated.

[0112] The “therapeutic effective dose” or “preventive effective dose” also includes the amount of RNAi agent that produces some desired local or systemic effect with a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0113] As used herein, the terms “therapeutic effective dose” and “preventive effective dose” also include amounts that provide benefit in the treatment, prevention, or management of a pathological process or symptoms of a pathological process mediated by TTR expression. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, hypoesthesia in the distal extremities), autonomic neuropathy (e.g., gastrointestinal dysfunction, e.g., gastric ulcers, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial neuropathy, and lattice corneal dystrophy.

[0114] In one embodiment, for example, when a subject has FAP, mixed-phenotypic FAP, mixed-phenotypic FAC, or FAP and OLT, treatment of the subject with the dsRNA agent of the present invention slows the progression of neuropathy. In another embodiment, for example, when a subject has FAP, mixed-phenotypic FAP, mixed-phenotypic FAC, SSA, or FAP and OLT, treatment of the subject with the dsRNA agent of the present invention slows the progression of neuropathy and cardiomyopathy. In yet another embodiment, for example, when a subject has cardiac impairment, the method of the present invention improves the structure and function of the heart, for example, by reducing mean left ventricular wall thickness and longitudinal strain, and by lowering the expression level of N-terminal pro-β natriuretic peptide (NT-proBNP), a cardiac stress biomarker.

[0115] Administration of the RNAi agent of the present invention in therapeutic or prophylactic effective doses is also useful in methods for improving neurological impairment and / or at least one indicator of quality of life in subjects suffering from or at risk of developing TTR-related diseases.

[0116] For example, in one embodiment, the method of the present invention improves at least one indicator of neurological impairment in a subject. “Improving at least one indicator of neurological impairment” in a subject means the ability of the method of the present invention to delay, reduce, or halt neurological impairment, or to improve any symptoms associated with neurological impairment. Any preferred measure of neurological impairment can be used to determine whether the subject has reduced, delayed, or halted neurological impairment, or whether there has been improvement in symptoms associated with neurological impairment.

[0117] One suitable measure is the Neuropathy Score (NIS). The NIS refers to a scoring system that assesses muscle weakness, sensation, and reflexes, particularly in relation to peripheral neuropathy. The NIS score assesses standard muscle groups for muscle weakness (1 is a 25% decrease, 2 is a 50% decrease, 3 is a 75% decrease, 3.25 is movement against gravity, 3.5 is movement under excluded gravity, 3.75 is muscle spasm without movement, and 4 is paralysis), standard muscle stretch reflexes (0 is normal, 1 is low, 2 is absent), as well as touch pressure, vibration, joint position and movement, and pin pain sensation (all graded for the index finger and big toe: 0 is normal, 1 is low, 2 is absent). The assessment is adjusted for age, sex, and physical fitness.

[0118] In one embodiment, the method of the present invention reduces the NIS by at least 10%. In another embodiment, the method of the present invention results in a reduction of at least 5, 10, 15, 20, 25, 30, 40, or at least 50% of the NIS. In yet another embodiment, the method stops the increasing NIS score, for example, resulting in a 0% increase in the NIS score. In yet another embodiment, the method of the present invention slows the rate at which the NIS score increases, for example, the rate at which the NIS score increases in a subject treated with the RNAi agent of the present invention, compared to the rate at which the NIS score increases in a subject not treated with the RNAi agent of the present invention.

[0119] Methods for determining NIS in human subjects are well known to those skilled in the art and can be found, for example, in Dyck, PJ et al., (1997) Neurology 1997.49(1):pgs.229-239); Dyck PJ. (1988) Muscle Nerve. Jan;11(l):21-32.

[0120] Another preferred measure of neuropathy is the revised neuropathy score (mNIS+7). As is known to those skilled in the art, mNIS+7 refers to the clinically based assessment (NIS) of neuropathy combined with electrophysiological measures of small and large nerve fiber function (NCS and QST), as well as measures of autonomic function (postural blood pressure). The mNIS+7 score is a revision of the NIS+7 score (representing NIS+7 tests). In NIS+7, muscle weakness and muscle stretch reflexes are analyzed. Five of the seven tests include nerve conduction attributes. These attributes are peroneal complex muscle action potential amplitude, motor nerve conduction velocity and motor nerve distal latency (MNDL), tibial MNDL, and sural sensory nerve action potential amplitude. These values ​​are corrected for variables of age, sex, height, and weight. The remaining two of the seven tests include vibration detection threshold and heart rate reduction with deep breathing.

[0121] The mNIS+7 score is a revised version of NIS+7 that incorporates the use of Smart Somatotopic Quantitative Sensation Testing as a new autonomic assessment, as well as the use of compound muscle action potentials of the ulnar, fibular, and tibial nerves, and sensory nerve action potentials of the ulnar and sural nerves (Suanprasert, N. et al., (2014) J. Neurol. Sci., 344(1-2):pgs.121-128).

[0122] In one embodiment, the method of the present invention reduces the mNIS+7 score by at least 10%. In another embodiment, the method of the present invention results in a reduction of at least 5, 10, 15, 20, 25, 30, 40, or at least 50% of the mNIS+7 score. In yet another embodiment, the method stops the increasing mNIS+7, for example, resulting in a 0% increase in mNIS+7. In yet another embodiment, the method of the present invention slows the rate at which the NIS+7 score increases, for example, the rate at which the NIS+7 score increases in subjects treated with the RNAi agent of the present invention, compared to the rate at which the NIS+7 score increases in subjects not treated with the RNAi agent of the present invention.

[0123] In another embodiment, the method of the present invention improves at least one indicator of quality of life in a subject. “Improving at least one indicator of quality of life” in a subject means the ability of the method of the present invention to delay, reduce, or halt the deterioration of quality of life, or to improve quality of life. Any suitable measure of quality of life can be used to determine whether the subject is reducing, delaying, or halting the deterioration of quality of life, or improving quality of life.

[0124] For example, the SF-36® Health Survey provides a self-report, multi-item scale that measures eight health parameters: physical functioning, role limitations due to physical health problems, physical pain, general health, vitality (energy and fatigue), social functioning, role limitations due to emotional problems, and mental health (psychological distress and mental well-being). The survey also provides a physical component summary and a mental component summary.

[0125] In one embodiment, the method of the present invention targets improvement over baseline in at least one of the SF-36 physical health-related parameters (physical health, role-physical, bodily pain, and / or general health) and / or at least one of the SF-36 mental health-related parameters (vitality, social functioning, role-emotional, and / or mental health). Such improvement may take the form of an increase of at least one, for example, at least two or at least three points on a scale for any one or more parameters.

[0126] In other embodiments, the method of the present invention stops the decreasing SF-36 parameter score for any one or more parameters, for example, the method results in a 0% decrease in SF-36. In yet another embodiment, the method of the present invention slows the rate at which the SF-36 score decreases, for example, the rate at which the SF-36 score decreases in subjects treated with the RNAi agent of the present invention, compared to the rate at which the SF-36 score decreases in subjects not treated with the RNAi agent of the present invention.

[0127] Another preferred measure of quality of life is the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) questionnaire. The Norfolk QOL-DN is a verified, comprehensive questionnaire designed to capture the full spectrum of DN, including large-diameter fibers, small-diameter fibers, and autonomic dysfunctions that are not captured by existing devices.

[0128] In one embodiment, the method of the present invention improves the Norfolk QOL-DN score of a subject from baseline, for example, by a change of about -2.5, -3.0, -3.5, -4.0, -4.5, -5.0, -5.5, -6.0, -6.7, -7.0, -7.5, -8.0, -8.5, -9.0, -9.5, or about -10.0. In another embodiment, the method stops the increasing Norfolk QOL-DN score, for example, resulting in a 0% decrease in the Norfolk QOL-DN score. In yet another embodiment, the method of the present invention slows the rate at which the QOL-DN score increases, for example, the rate at which the QOL-DN score increases in a subject treated with the RNAi agent of the present invention, compared to the rate at which the QOL-DN score increases in a subject not treated with the RNAi agent of the present invention.

[0129] Another suitable measure of quality of life is exercise intensity, such as that assessed by the NIS-W score. The NIS-W score is a composite score that sums up muscle weakness in the head, trunk, and limbs. Using NIS(W) (referring to a part of the scale that measures muscle weakness), muscle strength is assessed as normal (0) or complete paralysis (4), with a score of 1 representing an intermediate grade; a muscle considered to have a 25% decrease by clinical strength testing; 2 as a 50% decrease; 3 as a 75% decrease; 3.25 as movement against gravity; 3.50 as movement under excluded gravity; and 3.75 as muscle twitching.

[0130] In one embodiment, the method of the present invention provides an improvement to the baseline in the NIS-W score. Such improvement may take the form of an increase of at least 1, for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 points in the subject's NIS-W score, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 points. In another embodiment, the method stops the decline in the NIS-W score, for example, the method results in a 0% decrease in the NIS-W score. In yet another embodiment, the method of the present invention slows the rate at which the NIS-W score declines, for example, the rate at which the NIS-W score declines in a subject treated with the RNAi agent of the present invention, compared to the rate at which the NIS-W score declines in a subject not treated with the RNAi agent of the present invention.

[0131] Another preferred indicator of quality of life is the Rasch-built Overall Disability Scale (R-ODS), a patient questionnaire designed to capture limitations in activity and social participation in patients. In one embodiment, the method of the present invention provides an improvement over baseline in the R-ODS score. Such improvement may take the form of an increase of at least 0.1, e.g., at least 0.2, at least 0.3, at least 0.4, or at least 0.5, e.g., 0.1, 0.2, 0.3, 0.4, or 0.5 points in the subject's R-ODS score. In another embodiment, the method halts the decline in the R-ODS score, for example, resulting in a 0% decrease in the R-ODS score. In yet another embodiment, the method of the present invention slows the rate at which the R-ODS score declines, e.g., the rate at which the R-ODS score declines in subjects treated with the RNAi agent of the present invention compared to the rate at which the R-ODS score declines in subjects not treated with the RNAi agent of the present invention.

[0132] The Composite Autonomic Symptom Score (COMPASS-31) is a patient questionnaire used to assess symptoms of autonomic dysfunction and is another preferred indicator of quality of life. In one embodiment, the method of the present invention provides an improvement over baseline in the COMPASS-31 score. Such improvement may take the form of an increase of at least 0.1, e.g., at least 0.2, at least 0.3, at least 0.4, or at least 0.5, e.g., 0.1, 0.2, 0.3, 0.4, or 0.5 points in the subject's COMPASS-31 score. In another embodiment, the method halts the decline in the COMPASS-31 score, for example, resulting in a 0% decrease in the COMPASS-31 score. In yet another embodiment, the method of the present invention slows the rate at which the COMPASS-31 score declines, e.g., the rate at which the COMPASS-31 score declines in a subject treated with the RNAi agent of the present invention compared to the rate at which the COMPASS-31 score declines in a subject not treated with the RNAi agent of the present invention.

[0133] Other quality of life indicators may include nutritional status, for example, assessed by a change in median body mass index (mBMI). In one embodiment, the method of the present invention targets an improvement in mBMI relative to baseline. Such improvement may take the form of a decrease of approximately 2, 5, 7, 10, 12, 15, 20, or approximately 25 in the mBMI score. In another embodiment, the method stops the increasing mBMI index score, for example, resulting in a 0% increase in the mBMI score. In yet another embodiment, the method of the present invention slows the rate at which the mBMI score increases, for example, the rate at which the mBMI score increases in subjects treated with the RNAi agent of the present invention, compared to the rate at which the mBMI score increases in subjects not treated with the RNAi agent of the present invention.

[0134] Another quality of life indicator includes the assessment of motor capacity. One preferred measure of motor capacity is the 6-minute walk test (6MWT), which measures how far a subject can walk in 6 minutes, i.e., the 6-minute walk distance (6MWD). In one embodiment, the method of the present invention provides for subjects with an increase of at least about 10 minutes from baseline in 6MWD, for example, about 10, 15, 20, or about 30 minutes.

[0135] Another preferred measure is a 10-meter walk test to measure walking speed. In one embodiment, the method of the present invention provides for an increase of at least about 10 minutes from baseline in a 10-meter walk test, for example, about 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or about 5.0 m / sec.

[0136] The methods of the present invention may also improve the prognosis of the subject being treated. For example, the methods of the present invention may provide a reduction in the probability of clinical deterioration events during the treatment period, and / or increased life expectancy, and / or reduced hospitalizations.

[0137] The dose of RNAi agent administered to the subject may be adjusted to balance the risks and benefits of a particular dose, thereby achieving, for example, the desired level of TTR gene suppression (e.g., as an assessment based on TTR mRNA suppression, TTR protein expression, or reduction of amyloid deposition, as defined above) or the desired therapeutic or prophylactic effect, while simultaneously avoiding undesirable side effects.

[0138] In one embodiment, the iRNA agent of the present invention is administered to a subject in a body weight-based dose. A "body weight-based dose" (e.g., a dose in mg / kg units) is a dose of the iRNA agent that will vary depending on the subject's body weight. In another embodiment, the iRNA agent is administered to a subject in a constant dose. A "constant dose" (e.g., a dose in mg units) means that one dose of the iRNA agent is used for all subjects, regardless of any particular subject-specific factor, such as body weight. In a particular embodiment, the constant dose of the iRNA agent of the present invention is based on a predetermined body weight or age.

[0139] In some embodiments, the RNAi agent is administered as a fixed dose between approximately 15 mg and approximately 100 mg, for example, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 45 mg, approximately 50 mg, approximately 55 mg, approximately 60 mg, approximately 65 mg, approximately 70 mg, approximately 75 mg, approximately 80 mg, approximately 85 mg, approximately 90 mg, approximately 95 mg, or approximately 100 mg.

[0140] In one embodiment, the RNAi agent is administered to the subject at a constant dose of approximately 15, 20, 25, 30, 35, 40, 45, or approximately 50 mg once every three months (i.e., once every quarter). In another embodiment, the RNAi agent is administered to the subject at a constant dose of approximately 15, 20, 25, 30, 35, 40, 45, or approximately 50 mg once every four months. In yet another embodiment, the RNAi agent is administered to the subject at a constant dose of approximately 15, 20, 25, 30, 35, 40, 45, or approximately 50 mg once every five months. In yet another embodiment, the RNAi agent is administered to the subject at a constant dose of approximately 15, 20, 25, 30, 35, 40, 45, or approximately 50 mg once every six months. In one embodiment, administration is subcutaneous, e.g., self-administration, e.g., via a pre-filled syringe or an auto-injector syringe. In some embodiments, the dose of the RNAi agent for subcutaneous administration is contained in a volume of, for example, 1 ml or less of a pharmaceutically acceptable carrier.

[0141] In some embodiments, the RNAi agent is administered in two or more doses. If it is desirable to facilitate repeated or frequent infusions, implantation of a delivery device, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, cisterna magna, or intra-articular), or a reservoir may be advisable. In some embodiments, the number or amount of subsequent doses depends on achieving the desired effect, e.g., suppression of the TTR gene, or achieving a therapeutic or preventive effect, e.g., reducing amyloid deposition or reducing the symptoms of TTR-related disease.

[0142] In some embodiments, the RNAi agent is administered according to a schedule. For example, the RNAi agent may be administered twice, three times, four times, or five times per week. In some embodiments, the schedule includes administration at regular intervals. In other embodiments, the schedule includes administrations at close intervals followed by administrations at longer intervals (during which the active ingredient is not administered). In certain embodiments, the longer interval is determined based on increasing over time or the achievement of the desired effect.

[0143] Any of these schedules may be optionally repeated over one or more iterations. The number of iterations may depend on achieving the desired effect, e.g., suppression of the TTR gene, retinol-binding protein levels, vitamin A levels, and / or achieving a therapeutic or preventive effect, e.g., reducing amyloid deposition or reducing symptoms of TTR-related disease.

[0144] In some embodiments, RNAi agents are administered in conjunction with other therapeutic agents or other treatment plans. For example, other agents or other treatment plans suitable for treating TTR-related diseases may include liver transplants, heart transplants, pacemaker implantation, and agents that can reduce mutant TTR levels in the body; tafamidis (INN, or Fx-1006A or vyndaqel) which kinetically stabilizes TTR tetramers by preventing the dissociation of tetramers required for TTR amyloid formation; nonsteroidal anti-inflammatory drugs (NSAIDs), such as diflunisal, which may be used to reduce edema in TTR amyloidosis, for example, in conjunction with cardiac disorders, and diuretics.

[0145] In one embodiment, the subject is administered an RNAi agent in an initial dose and one or more maintenance doses. The one or more maintenance doses may be less than or equal to the initial dose, for example, half of the initial dose. Furthermore, the treatment plan may extend over a period of time that will vary depending on the nature of the specific disease, its severity, and the patient's overall condition. In certain embodiments, the dose may be delivered no more than once per day, for example, once every 24, 36, 48, or no more than once per longer period, for example, once every 5 or 8 days. After treatment, the patient may be monitored for changes in his / her condition. The dose of the RNAi agent may be increased if the patient does not respond significantly to the current dose level, or the dose may be decreased if there is a reduction in the symptoms of the condition, if the condition has disappeared, or if unwanted side effects are observed.

[0146] In some embodiments of the method of the present invention, TTR gene expression is inhibited to at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or below the detection level of the assay. In some embodiments, inhibition of TTR gene expression results in normalization of TTR gene levels such that the difference between pre-treatment levels and normal control levels is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the inhibition is clinically relevant.

[0147] The term “inhibit,” as used herein, is interchangeable with “reduce,” “deactivate,” “downregulate,” “suppress,” and other similar terms, and includes any level of inhibition. Preferably, inhibit includes statistically significant or clinically significant inhibition.

[0148] The phrase "inhibit TTR expression" is intended to refer to the inhibition of expression of any TTR gene (e.g., mouse TTR gene, rat TTR gene, monkey TTR gene, or human TTR gene), and variants or mutants of the TTR gene. Therefore, the TTR gene may be a wild-type TTR gene, a mutant TTR gene (e.g., a mutant TTR gene resulting in amyloid deposition), or a transgenic TTR gene in relation to genetically modified cells, cell populations, or organisms.

[0149] "Inhibiting TTR gene expression" includes inhibition of TTR gene expression at any level, e.g., at least partial suppression of TTR gene expression. TTR gene expression may be assessed based on the level or change at that level of any variable associated with TTR gene expression, e.g., TTR mRNA level, TTR protein level, or the number or extent of amyloid deposits. This level may be assessed in individual cells or cell populations, e.g., including a sample derived from the subject.

[0150] Inhibition may be assessed by a decrease in the absolute or relative level of one or more variables associated with TTR expression compared to a control level. The control level may be any type of control level used in the art, e.g., a pre-dosing baseline level, or a level determined from similar subjects, cells, or samples that are not treated with or are treated with a control (e.g., a buffer-only control or an inactivator control).

[0151] Inhibition of TTR gene expression may be indicated by a decrease in the amount of mRNA expressed by a first group of cells (such cells may be present, for example, in a sample derived from the subject) that have been treated (for example, by contacting one or more cells with the RNAi agent of the present invention, or by administering the RNAi agent of the present invention to a subject in which cells are present or have been present) so that the TTR gene is transcribed and TTR gene expression is inhibited, compared to a second group of cells (control cells) that are substantially identical to the first group of cells but have not been treated in the same way. In a preferred embodiment, the inhibition is expressed by the following formula:

number

[0152] Alternatively, inhibition of TTR gene expression may be evaluated in terms of a decrease in TTR gene expression, e.g., TTR protein expression, retinol-binding protein levels, vitamin A levels, or parameters functionally related to the presence of amyloid deposition containing TTR. TTR gene silencing may be determined constitutively or by genomic engineering in any cell expressing TTR, and by any assay known in the art. The liver is a major site of TTR expression. Other prominent expression sites include the choroid plexus, retina, and pancreas.

[0153] Inhibition of TTR protein expression may be indicated by a decrease in the level of TTR protein expressed by cells or cell populations (e.g., the level of protein expressed in a sample derived from the target). As described above for the evaluation of mRNA suppression, inhibition of protein expression levels in treated cells or cell populations may also be expressed as a percentage of the protein level in control cells or cell populations.

[0154] Control cells or cell populations that may be used to evaluate the inhibition of TTR gene expression include cells or cell populations that have not yet been exposed to the RNAi agent of the present invention. For example, control cells or cell populations may be derived from an individual subject (e.g., a human or animal subject) prior to treatment with the RNAi agent of the present invention.

[0155] The level of TTR mRNA expressed by a cell or cell population, or the level of circulating TTR mRNA, may be measured using any method known in the art for evaluating mRNA expression. In one embodiment, the level of TTR expression in a sample is measured by detecting transcribed polynucleotides, or a portion thereof, such as the mRNA of the TTR gene. RNA may be extracted from cells using RNA extraction techniques, for example, phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay forms utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, ribonuclease protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in-situ hybridization, and microarray analysis. Circulating TTR mRNA may be detected using the method described in international application PCT / US2012 / 043584 (the entire content of which is incorporated herein by reference).

[0156] In one embodiment, the expression level of TTR is measured using a nucleic acid probe. The term “probe,” as used herein, refers to any molecule capable of selectively binding to a particular TTR. Probes may be synthesized by those skilled in the art or derived from suitable biological preparations. Probes may be designed specifically for labeling. Examples of molecules available as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0157] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern blot analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for measuring mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with TTR mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In other embodiments, for example, in an Affymetrix gene chip array, the probe is immobilized on a solid surface and the mRNA is contacted with the probe. Those skilled in the art can easily adapt known mRNA detection methods to applications such as measuring TTR mRNA levels.

[0158] Alternative methods for measuring TTR expression levels in a sample include nucleic acid amplification (for cDNA preparation) of mRNA in the sample and / or reverse transcriptase, e.g., RT-PCR (experimental embodiments shown in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-persistent sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. The invention includes a process of detecting the amplified molecule by any nucleic acid amplification method (U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by a process of detecting the amplified molecule using techniques well known to those skilled in the art. These detection schemes are particularly useful in detecting nucleic acid molecules when such molecules are present in very small numbers. In certain embodiments of the invention, the level of TTR expression is measured by quantitative fluorescence-generating RT-PCR (i.e., the TaqMan® system).

[0159] The expression level of TTR mRNA may be monitored using membrane blotting (e.g., used in hybridization analyses such as Northern, Southern, and Dot), or using microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934 (incorporated herein by reference). Measurement of TTR expression levels may also include the use of nucleic acid probes in solution.

[0160] In preferred embodiments, mRNA expression levels are assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of these methods is described and illustrated in the examples presented herein.

[0161] The level of TTR protein expression may be measured using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assays, and electrochemiluminescence assays.

[0162] In some embodiments, the effectiveness of the methods of the present invention may be monitored by detecting or monitoring a reduction in amyloid-TTR deposition. Reducing amyloid-TTR deposition includes any reduction in the size, number, or severity of TTR deposition, or inhibition or reduction in the formation of TTR deposition within the organ or region of interest, when used herein, which may be evaluated in vitro or in vivo using any method known in the art. For example, several methods for evaluating amyloid deposition are described in Gertz, MA & Rajukumar, SV (Editors) (2010), Amyloidosis: Diagnosis and Treatment, New York: Humana Press. Methods for evaluating amyloid deposition may include biochemical analysis, as well as visual or computerized evaluation of amyloid deposition when visualized using, for example, immunohistochemical staining, fluorescence labeling, light microscopy, electron microscopy, fluorescence microscopy, or other types of microscopy. To evaluate amyloid deposition, invasive or non-invasive imaging methods may be used, including, for example, CT, PET, or NMR / MRI imaging.

[0163] The methods of the present invention may reduce TTR deposition in several tissues or regions of the body, including, but are not limited to, the heart, liver, spleen, esophagus, stomach, intestines (ileum, duodenum, and colon), brain, sciatic nerve, dorsal root ganglia, kidneys, and retina.

[0164] The term “sample,” as used herein, includes collections of similar bodily fluids, cells, or tissues isolated from a subject, as well as bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples derived from tissue, organ, or local area. For example, a sample may be derived from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may be derived from the liver (e.g., the whole liver or a specific segment of the liver or a specific type of cell in the liver, e.g., hepatocytes), the retina or a part of the retina (e.g., retinal pigment epithelium), the central nervous system or a part of the central nervous system (e.g., the ventricles or choroid plexus), or the pancreas or specific cells or parts of the pancreas. In preferred embodiments, “sample derived from subject” means blood, or plasma or serum obtained from blood collected from the subject. In further embodiments, “sample derived from subject” refers to liver tissue (or a subcomponent thereof) or blood tissue (or a subcomponent thereof, e.g., serum) derived from subject.

[0165] In some embodiments of the method of the present invention, the RNAi agent is administered to a subject so that the RNAi agent is delivered to a specific site within the subject. Inhibition of TTR expression may be evaluated by measuring the level or change in level of TTR mRNA or TTR protein in a sample derived from body fluid or tissue from a specific site within the subject. In preferred embodiments, the site is selected from the group consisting of the liver, choroid plexus, retina, and pancreas. The site may also be a subsection or subgroup of cells (e.g., hepatocytes or retinal pigment epithelium) from any one of the above sites. The site may also include cells expressing a specific type of receptor (e.g., hepatocytes expressing the asialoglycoprotein receptor).

[0166] III. iRNA of the present invention iRNAs suitable for use in the methods of the present invention include double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the TTR gene in cells, e.g., cells inside a subject, e.g., a mammal, e.g., a human having a TTR-related disease. The dsRNA includes an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during TTR gene expression. The complementary region is approximately 30 nucleotides or less in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less). Upon contact with cells expressing 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 approximately 10% in assays, e.g., by PCR or branched DNA (bDNA) based methods, or by protein-based methods such as immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0167] A dsRNA contains two complementary RNA strands, which hybridize under the conditions in which the dsRNA is used to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary to the target sequence, and generally fully complementary. The target sequence may originate from the mRNA sequence formed during the expression of a TTR gene. The other strand (the sense strand) contains 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 in this specification and as known in the art, the complementary sequence of a dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to that on a separate oligonucleotide.

[0168] Generally, double-stranded structures are, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 These range from 15 to 30 base pairs in length, such as 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths beyond the listed ranges and lengths are also intended to be part of the present invention.

[0169] Similarly, complementary regions of target sequences are, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-2 7, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide lengths, etc. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0170] In some embodiments, the dsRNA is about 15–20 nucleotides long, or about 25–30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. It is well known in the art that dsRNA longer than about 21–23 nucleotides may serve as a substrate for Dicer. As those skilled in the art will recognize, 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 “part” of the mRNA target is a continuous sequence of mRNA targets long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0171] Those skilled in the art can, for example, use approximately 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-3 3, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-1 9, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20 It will also be recognized that double-stranded regions of approximately 9 to 36 base pairs, such as ~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29, 21~28, 21~27, 21~26, 21~25, 21~24, 21~23, or 21~22 base pairs, are the main functional parts of dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs, within the range that it is processed into a functional double-strand of 15 to 30 base pairs that targets a desired RNA for cleavage, is a dsRNA. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a native miRNA. In another embodiment, iRNA agents useful for targeting TTR expression are not generated in the target cell by cleaving larger dsRNAs.

[0172] The dsRNAs described herein may further comprise one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit surprisingly superior inhibitory properties compared to their blunt-end equivalents. Nucleotide overhangs may comprise, or consist of, nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. Overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA. In certain embodiments, longer, extended overhangs are possible.

[0173] dsRNA can be synthesized by standard methods known in the art, using automated DNA synthesizers, such as those commercially available from Biosearch, Applied Biosystems, Inc., as will be further discussed below.

[0174] The iRNA compounds of the present invention may be prepared using a two-step method. First, the individual strands of a double-stranded RNA molecule are prepared separately. Next, the constituent strands are annealed. The individual strands of the siRNA compound can be prepared using solution phase, solid-phase organic synthesis, or both. Organic synthesis offers the advantage of easily preparing oligonucleotide chains containing non-natural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase, solid-phase organic synthesis, or both.

[0175] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences: a sense sequence and an antisense sequence. The sense strand is selected from the sequence group provided in Table 1, and the antisense strand corresponding to the sense strand is selected from the sequence group in Table 1. In this embodiment, one of the two sequences is complementary to the other, and one of the sequences is substantially complementary to the mRNA sequence that occurs during TTR gene expression. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is described as the sense strand in Table 1, and the second oligonucleotide is described as the antisense strand corresponding to the sense strand in Table 1. 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.

[0176] Some of the sequences in Table 1 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 listed in Table 1, which may be unmodified, unconjugated, and / or modified and / or conjugated in a manner different from those described.

[0177] Those skilled in the art are well aware that dsRNAs having double-stranded structures of approximately 20–23 base pairs, such as 21 base pairs, are supported as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877–6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures may also be equally effective (Chu and Rana (2007) RNA 14:1714–1719; Kim et al. (2005) Nat Biotech 23:222–226). In the embodiments described above, due to the nature of the oligonucleotide sequences provided in Table 1, the dsRNAs described herein may comprise at least one strand of a minimum length of 21 nucleotides. It can be reasonably expected that shorter double-stranded structures having one of the sequences in Table 1, with only a few nucleotides missing from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, or 20 or more consecutive nucleotides derived from one of the sequences in Table 1, and having the ability to inhibit TTR gene expression, are intended to be within the scope of the present invention if they are different from dsRNAs containing a full-length sequence, with approximately 5, 10, 15, 20, 25, or 30% of the sequence.

[0178] Furthermore, the RNAs provided in Table 1 identify sites in TTR transcripts that are highly sensitive to RISC-mediated cleavage. Therefore, the present invention further features iRNAs that target within one of these sequences. In the use herein, an iRNA is said to target a specific site within the RNA transcript if it promotes cleavage of the transcript somewhere within that site. Such iRNAs generally consist of approximately 15 consecutive nucleotides from one of the sequences provided in Table 1, ligated to an additional nucleotide sequence from a region adjacent to a selected sequence in the TTR gene.

[0179] Target sequences are generally about 15–30 nucleotides long, but there is a wide range of variation 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 the optimal target sequence for any given gene target, but an empirical approach can also be taken to identify sequences within a size range that could act as the target sequence by actually or figuratively (including, for example, by computer simulation) placing a “window” or “mask” of a given size (21 nucleotides as an unrestricted example) on the target RNA sequence. By sequentially moving the sequence “window” one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing to identify optimally functioning sequences (using assays described herein or known in the art), can identify the RNA sequence that best mediates the inhibition of target gene expression when targeted with an iRNA agent. Therefore, while the sequences identified in Table 1, for example, represent effective target sequences, it is possible to further optimize inhibition efficiency by identifying sequences with equivalent or better inhibitory properties by sequentially "walking a window" one nucleotide upstream or downstream of a given sequence.

[0180] Furthermore, it is explored that further optimization of any sequence identified in Table 1, for example, can be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking a window of size longer or shorter than the target RNA from that position. Again, combining this approach to creating new target candidates with testing the efficacy of iRNAs based on these target sequences in inhibitory assays known in the art and / or described herein may lead to further improvements in inhibitory efficiency. Moreover, such optimized sequences can be modified by further optimizing the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting specific sites or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or considered herein.

[0181] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA drug chain complementary to the TTR gene region, the RNA chain generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it may be determined whether an iRNA containing a mismatch with the target sequence is effective in inhibiting TTR gene expression. Examining the effectiveness of mismatched iRNAs in inhibiting TTR gene expression is important, especially when a particular complementary region of the TTR gene is known to have polymorphic sequence variations within the population.

[0182] IV. Modified iRNA of the present invention In one embodiment, the RNA of the iRNA intended for use in the method of the present invention, e.g., dsRNA, is unmodified and does not contain, for example, chemical modifications and / or conjugations known in the art and described herein. In another embodiment, the RNA of the iRNA agent intended for use in the method of the present invention, e.g., dsRNA, is chemically modified to enhance stability or other advantageous characteristics. In certain embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all 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 includes 8 or fewer 2'-fluoro modifications on the sense strand (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, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications) and 6 or fewer 2'-fluoro modifications on the antisense strand (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). In other embodiments, all nucleotides of the iRNA of the present invention are modified, and the iRNA includes 8 or fewer 2'-fluoro modifications on the sense strand (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, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications) and 6 or fewer 2'-fluoro modifications on the antisense strand (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). In the case where "substantially all nucleotides are modified," the iRNA of the present invention is extensively modified, though not entirely, and may contain 5 or fewer, 4, 3, 2, or 1 unmodified nucleotides.

[0183] Nucleic acids discussed herein can be synthesized and / or modified by methods established in the art, such as those described herein by reference in “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Examples of modifications include terminal modifications such as 5'-end modifications (phosphorylation, conjugation, inversion) or 3'-end modifications (conjugation, DNA nucleotide, inversion); base modifications such as substitution, base removal (debasing nucleotide), or conjugated bases, whether at a stabilizing base, a destabilizing base, or a base that forms a base pair with an expanding partner repertoire; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and main chain modifications, including modification or substitution of phosphate diester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNA containing a modified main chain or RNA without natural nucleoside interbonding. RNAs having a modified backbone include those that do not have a phosphorus atom in their backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone are also considered oligonucleosides. In some embodiments, modified iRNAs have a phosphorus atom in their internucleoside backbone.

[0184] Examples of modified RNA backbone include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylenephosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages and their 2'-5' linked analogues, as well as boranophosphates with reversed polarity where adjacent nucleoside unit pairs are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0185] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above are 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. 5,286,7 Specification No. 17; US Patent Nos. 5,321,131; US ​​Patent Nos. 5,399,676; US Patent Nos. 5,405,939; US Patent Nos. 5,453,496; US Patent Nos. 5,455,233; US Patent Nos. 5,466,677; US Patent Nos. 5,476,925; US Patent Nos. 5,519,126; US Patent Nos. 5,536,821; US ​​Patent Nos. 5,541,316; US Patent Nos. 5,550,111; US ​​Patent Nos. 5,563,253; US Patent Nos. 5,57 U.S. Patent No. 1,799; 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 U.S. Patent Nos. 531,590; U.S. Patent Nos. 6,534,639; U.S. Patent Nos. 6,608,035; U.S. Patent Nos. 6,683,167; U.S. Patent Nos. 6,858,715; U.S. Patent Nos. 6,867,294; U.S. Patent Nos. 6,878,805; U.S. Patent Nos. 7,015,315; U.S. Patent Nos. 7,041,816; U.S. Patent Nos. 7,273,933; U.S. Patent Nos. 7,321,029; and U.S. Patent No. RE39464, but not limited to these.

[0186] Modified RNA backchains that do not contain a phosphorus atom have backchains formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar portion of nucleosides); siloxane backchains; sulfide, sulfoxide, and sulfone backchains; formacetyl and thioformacetyl backchains; methyleneformacetyl and thioformacetyl backchains; alkene-containing backchains; sulfamate backchains; methyleneimino and methylenehydrazino backchains; sulfonate and sulfonamide backchains; those having amide backchains; and others having mixed N, O, S, and CH2 components.

[0187] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides are U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967, all of which are incorporated herein by reference in their entirety. Examples of U.S. Patent Nos. include, but are not limited to, U.S. Patent Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439.

[0188] In another embodiment, a suitable RNA mimetic is considered for use in iRNA, in which both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with a new group. The base units are maintained for hybridization with a suitable nucleic acid target compound. Such an oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bind directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, each of which is incorporated herein by reference in its entirety. Furthermore, PNA compounds suitable for use in iRNA according to the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0189] Some embodiments of the present invention include RNA having a phosphorothioate backbone, and oligonucleosides having a heteroatom backbone which is --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [natural phosphate diester backbone is represented as --O--P--O--CH2--], and an amide backbone as described in U.S. Patent No. 5,602,240. In some embodiments, the RNA discussed herein has the morpholino backbone structure described in the aforementioned U.S. Patent No. 5,034,506.

[0190] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs such as the dsRNAs discussed herein can include 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 1 to about 10). In another embodiment, the dsRNA includes 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 cleaving 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., alkoxy-alkoxy groups. Other exemplary modifications are 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described below in the examples herein.

[0191] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on 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 sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of the above-mentioned modified sugar structures include, specifically, 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, U.S. Patent Nos. 567,811; U.S. Patent Nos. 5,576,427; U.S. Patent Nos. 5,591,722; U.S. Patent Nos. 5,597,909; U.S. Patent Nos. 5,610,300; U.S. Patent Nos. 5,627,053; U.S. Patent Nos. 5,639,873; U.S. Patent Nos. 5,646,265; U.S. Patent Nos. 5,658,873; U.S. Patent Nos. 5,670,633; and U.S. Patent Nos. 5,700,920, among others. The entire contents of each of the above are incorporated herein by reference.

[0192] The RNA of the iRNA of the present invention may also include nucleic acid base (often simply referred to as "bases" in the art) modifications or substitutions. In the use herein, "unmodified" or "natural" nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include deoxythymine (dT), 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil) Other examples of synthetic and natural nucleic acid bases include 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 3-deazaguanine and 3-deazaadenine.Furthermore, examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, STand Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in this 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 double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276–278), making it an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0193] Representative U.S. patents teaching the preparation of the specific modified nucleic acid bases and other modified nucleic acid bases described above are, as their entire contents are incorporated herein by reference, U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469. U.S. Patent Nos. 5,594,121, 5,596,091; U.S. Patent Nos. 5,614,617; U.S. Patent Nos. 5,681,941; U.S. Patent Nos. 5,750,692; U.S. Patent Nos. 6,015,886; U.S. Patent Nos. 6,147,200; U.S. Patent Nos. 6,166,197; U.S. Patent Nos. 6,222,025; U.S. Patent Nos. 6,235,887; U.S. Patent Nos. 6,380,368; U.S. Patent Nos. 6,528,640; U.S. Patent Nos. 6,639,062; U.S. Patent Nos. 6,617,438; U.S. Patent Nos. 7,045,610; U.S. Patent Nos. 7,427,672; and U.S. Patent Nos. 7,495,088.

[0194] The RNA of iRNA can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge between two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of a sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the active ingredient 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 containing a bicyclic sugar moiety with 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 nonspecific effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides used in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides containing a 4'-to-2' bridge.Examples of such 4'-to-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' (also known as "bound ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 8 Examples include 4'-CH2-N(OCH3)-2' (and its analogues; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see, e.g., U.S. 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 analogues; see, e.g., U.S. Patent No. 8,278,426). The entirety of each of the foregoing is incorporated herein by reference.

[0195] Additional representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following, namely, U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are hereby incorporated by reference herein.

[0196] 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).

[0197] 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".

[0198] The iRNA of the present invention may also comprise one or more “structurally restricted nucleotides” (“CRNs”). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable configuration and increases hybridization affinity to mRNA. The linker is long enough to position oxygen in an optimal position for stability and affinity, resulting in reduced puckering of the ribose ring.

[0199] Representative publications teaching the preparation of specific CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication International Publication No. 2013 / 036868 (the entire contents of each of these are incorporated herein by reference).

[0200] One or more nucleotides of the iRNA of the present invention may also include a hydroxymethyl-substituted nucleotide. The "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seconucleotide (also referred to as "unlocked nucleic acid" ("UNA") modification).

[0201] Representative U.S. publications teaching the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020 (the entire contents of each of these are incorporated herein by reference).

[0202] Potential stabilization 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-docosanoyluridine-3''-phosphate, and the reversed base dT (idT). These modifications are disclosed in International Publication No. 2011 / 005861.

[0203] Other nucleotide modifications of the iRNA of the present invention include 5' phosphate or 5' phosphate mimetic on the antisense strand of the RNAi agent, e.g., 5' terminal phosphate or phosphate mimetic. Suitable phosphate mimetics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, which is incorporated herein by reference in its entirety.

[0204] A. Modified iRNA containing the motif of the present invention In particular aspects of the present invention, the double-stranded RNAi agent intended for use in the method of the present invention includes, for example, chemical modifications as disclosed in U.S. Provisional Patent Application No. 61 / 561,710 filed on 18 November 2011, or International Application PCT / US2012 / 065691 filed on 16 November 2012 (the contents of each of these are incorporated herein by reference).

[0205] More specifically, it has been surprisingly discovered that the gene silencing activity of a double-stranded RNAi agent is significantly enhanced when the sense and antisense strands of the RNAi agent are modified to have one or more identical modification motifs on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent.

[0206] 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 comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be in the range of 12 to 30 nucleotides in length. For example, each strand may be between 14 to 30 nucleotides, 17 to 30 nucleotides, 25 to 30 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or between 21 and 23 nucleotides in length.

[0207] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "RNAi agent." The double-stranded region of the RNAi agent may be 12–30 nucleotide pairs long. For example, the double-stranded region may be between 14–30 nucleotide pairs, 17–30 nucleotide pairs, 27–30 nucleotide pairs, 17–23 nucleotide pairs, 17–21 nucleotide pairs, 17–19 nucleotide pairs, 19–25 nucleotide pairs, 19–23 nucleotide pairs, 19–21 nucleotide pairs, 21–25 nucleotide pairs, or 21–23 nucleotide pairs. In another example, the double-stranded region may be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide lengths.

[0208] 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 long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The overhang may result from one strand being longer than the other, or from two strands of equal length being twisted. The overhang may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be a different 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.

[0209] In one embodiment, each nucleotide within the overhang region of the RNAi agent may independently be a modified or unmodified nucleotide, for example, but not limited to 2'-sugar modifications, such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT may be an overhang sequence at any 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 a different sequence.

[0210] The 5'- or 3'-overhangs on 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 them, where the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located within the antisense strand. In one embodiment, this 3'-overhang is located within the sense strand.

[0211] RNAi agents 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. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Although we do not want to be constrained by theory, asymmetric blunt ends at the 5' end of the antisense strand and overhangs at the 3' end of the antisense strand are preferable for loading the guide strand into a RISC process.

[0212] In one embodiment, the RNAi agent comprises 21 nucleotide sense strands and 23 nucleotide antisense strands, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the RNAi agent is blunt, while the other end contains a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is located at the 3' end of the antisense strand.

[0213] When a two-nucleotide overhang is present at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the three nucleotide ends, where two of the three nucleotides are the overhang nucleotide and the third nucleotide is a pairing nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide bonds between the three nucleotide ends at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. In one embodiment, all nucleotides of the iRNA of the present invention are modified, and the iRNA includes 8 or fewer 2'-fluoro modifications on the sense strand (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, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications) and 6 or fewer 2'-fluoro modifications on the antisense strand (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). Optionally, the RNAi agent further includes a ligand (preferably GalNAc3).

[0214] In one embodiment, the sense strand of the RNAi agent contains at least one of three identical modification motifs on three consecutive nucleotides, where one of the motifs is located at a cleavage site within the sense strand.

[0215] In one embodiment, the antisense strand of the RNAi agent also contains at least one of three identical modification motifs on three consecutive nucleotides, where one of the motifs is located at or near a cleavage site within the antisense strand.

[0216] In RNAi agents having a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically located at positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs may be located 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 begins either from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide inside the double-stranded region from the 5' end of the antisense strand. The cleavage sites within the antisense strand may also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0217] The sense strand of an RNAi agent may have at least one identically modified motif on three consecutive nucleotides at a cleavage site; and the antisense strand may have at least one identically modified motif on three consecutive nucleotides at or near a cleavage site. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand may 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 duplication, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0218] In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides of a portion 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 non-bridging phosphate oxygen and / or one or both of one or more bridging phosphate oxygens; alterations of components of the ribose sugar, such as the 2'-hydroxyl of the ribose sugar; substantial substitution with "dephospho" linkers in the phosphate moiety; modification or substitution of natural bases; and substitution or modification of the ribose-phosphate backbone.

[0219] 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 target position in the nucleic acid, but often it will not. As an 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 double-stranded regions of the RNA, or only within single-stranded regions of the RNA. For example, phosphorothioate modification at non-bridging O positions may occur only at one or both termini, 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 termini. The 5' terminus may be phosphorylated.

[0220] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in single-stranded overhangs, e.g., in the 5' or 3' overhang, or in both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or part of the bases in the 3' or 5' overhang may be modified with modifications, for example, those described herein. Modifications may include, for example, the use of modifications known in the art at the 2' position of ribose sugars, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F), or 2'-O-methyl modifications in place of ribosaccharides in nucleic acid bases, and modifications at phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0221] In one embodiment, each residue in the sense and antisense chains is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. These chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0222] At least two distinct modifications are typically present on the sense and antisense chains. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, or other modifications.

[0223] In one embodiment, N a and / or N bThis includes alternating pattern modifications. The term “alternating motif,” as used herein, refers to a motif having one or more modifications, where each modification occurs on an alternating nucleotide of a single chain. Alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be “ABABABABABAB…”, “AABBAABBAABB…”, “AABAABAABAAB…”, “AAABAAABAAAB…”, “AAABBBAAABBB…”, or “ABCABCABCABC…”.

[0224] The types of modifications contained 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 modifications to every other nucleotide, may be the same, but each of the sense strand or antisense strand may be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0225] In one embodiment, the RNAi agent of the present invention includes a change in the modification pattern of alternating motifs on the sense strand relative to the modification pattern of alternating motifs on the antisense strand. The change may be such that the modifying groups of the nucleotides on the sense strand correspond to different modifying groups of the nucleotides on the antisense strand, and vice versa. For example, when the sense strand pairs with the antisense strand in a dsRNA double strand, within the double-stranded region, the alternating motifs in the sense strand may begin with "ABABAB" from 5'-3' of the strand, and the alternating motifs in the antisense strand may begin with "BABABA" from 5'-3' of the strand. As another example, within the double-stranded region, the alternating motifs in the sense strand may begin with "AABBAABB" from 5'-3' of the strand, and the alternating motifs in the antisense strand may begin with "BBAABBAA" from 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.

[0226] In one embodiment, the RNAi agent includes having a pattern of alternating 2'-O-methyl and 2'-F modification motifs on the first sense strand that is different from the pattern of alternating 2'-O-methyl and 2'-F modification motifs on the first antisense strand, i.e., 2'-O-methyl modified nucleotides on the sense strand base-pair with 2'-F modified nucleotides on the antisense strand, and vice versa. The sense strand may begin at position 1 with a 2'-F modification, and the antisense strand may begin at position 1 with a 2'-O-methyl modification.

[0227] The introduction of one or more motifs of three identical modifications onto three consecutive nucleotides in the sense and / or antisense strand disrupts the initial modification pattern present in the sense and / or antisense strand. This disruption of the modification pattern in the sense and / or antisense strand, by introducing one or more motifs of three identical modifications onto three consecutive nucleotides in the sense and / or antisense strand, unexpectedly enhances gene silencing activity against the target gene.

[0228] In one embodiment, when three identical modification motifs on three consecutive nucleotides are introduced into any of the chains, the modifications of nucleotides adjacent to the motif are different from the modifications of the motif. For example, part of the sequence containing the motif is "...N a YYYN b ..." where "Y" represents the modification of three identical modification motifs on three consecutive nucleotides, and "N a " and "N b This indicates a modification to a nucleotide adjacent to the motif "YYY", which is different from the modification of Y, and also N a and N b These can be the same or different modifications. Or, N a and / or N b This may or may not exist when a wing modification is present.

[0229] The RNAi agent may further contain at least one internucleotide linkage of phosphorothioate or methylphosphonic acid. The internucleotide linkage modification of phosphorothioate or methylphosphonic acid may occur on any nucleotide at any position 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 antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand and / or antisense strand; or the sense strand or 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 have variations with respect to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent contains 6 to 8 internucleotide links of phosphorothioate. In one embodiment, the antisense strand includes two phosphorothioate internucleotide bonds at its 5' end and two phosphorothioate internucleotide bonds at its 3' end, and the sense strand includes at least two phosphorothioate internucleotide bonds at either its 5' or 3' end.

[0230] In one embodiment, the RNAi includes nucleotide-nucleotide linkage modifications of phosphorothioate or methylphosphonic acid within the overhang region. For example, the overhang region may include two nucleotides having a nucleotide-nucleotide linkage of phosphorothioate or methylphosphonic acid between the two nucleotides. The nucleotide-nucleotide linkage modifications may also be provided to link the overhang nucleotides to the terminal paired nucleotides inside the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked through nucleotide-nucleotide linkages of phosphorothioate or methylphosphonic acid, and optionally, additional nucleotide-nucleotide linkages of phosphorothioate or methylphosphonic acid may be present to link the overhang nucleotides to the paired nucleotides adjacent to the overhang nucleotides. For example, there may be at least two nucleotide-nucleotide linkages of phosphorothioate between three terminal nucleotides, where two of the nucleotides are overhang nucleotides and the third is a paired nucleotide adjacent to the overhang nucleotides. These three terminal nucleotides may be located 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.

[0231] In one embodiment, the two nucleotide overhangs are located at the 3' end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. Optionally, the RNAi agent may further have two phosphorothioate internucleotide bonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0232] In one embodiment, the RNAi agent includes mismatches with the target, within the double helix, or in combination thereof. The mismatches may be located within the overhang region or within the double helix region. Base pairs may be ranked based on their tendency to promote dissociation or dissolution (for example, the simplest method is to examine the pairings on a case-by-case basis for the free energy of association or dissociation of a particular pairing, although similar or analogous analyses may also be used). In terms of promoting dissociation, A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, such as non-standard or non-standard pairings (as described elsewhere in this specification), are preferred over standard (A:T, A:U, G:C) pairings; and pairings containing universal bases are preferred over standard pairings.

[0233] In one embodiment, the RNAi agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs within the double-stranded region from the 5' end of the antisense strand, independently selected from the group of A:U, G:U, I:C, and mismatch pairs, such as non-standard or non-standard pairings or pairings containing universal bases, in order to promote the dissociation of the antisense strand at the 5' end of the double-stranded region.

[0234] In one embodiment, the nucleotide at position 1 within the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs within the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair within the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0235] In one embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX)iN b -YYY-N b -(ZZZ) j -N a -n q 3'(I) (In the formula, i and j are independently either 0 or 1; p and q are each independently between 0 and 6; each N a Each represents an oligonucleotide sequence containing 0 to 25 modified nucleotides independently, with each sequence containing at least two different modified nucleotides; each N b This independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q This independently represents an overhanging nucleotide; N b and Y do not have the same modifications; and XXX, YYY, and ZZZ each independently represent a single motif of three identical modifications on three consecutive nucleotides. It may also be represented by . Preferably, YYY are all 2'-F modified nucleotides.

[0236] In one embodiment, N a and / or N b This includes alternating modification patterns.

[0237] In one embodiment, the YYY motif occurs at or near a cleavage site on the sense strand. For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may occur at or near a cleavage site on the sense strand (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13), where the count begins at the first nucleotide from the 5' end, or optionally, at the first paired nucleotide inside the double-stranded region from the 5' end.

[0238] 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. Therefore, the sense chain is given by the following equation: 5'n p -N a -YYY-Nb -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) It can be represented by:

[0239] When the sense chain is represented by equation (Ib), N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0240] When the sense chain is represented by equation (Ic), N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0241] When the sense chain is represented by equation (Id), each N b This independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. a X, Y, and Z can independently represent oligonucleotide sequences containing 2–20, 2–15, or 2–10 modified nucleotides. Each of X, Y, and Z may be the same as or different from one another.

[0242] In another embodiment, i is 0 and j is 0, and the sense chain is given by the formula: 5'n p -N a -YYY-N a -n q 3'(Ia) It may also be represented by [this method].

[0243] When the sense chain is represented by equation (Ia), each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0244] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5'n q '-N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II) (In the formula, k and l are independently either 0 or 1; p' and q' are each independently between 0 and 6; each N a ' represents an oligonucleotide sequence containing 0 to 25 modified nucleotides independently, with each sequence containing at least two different modified nucleotides; each N b ' represents an oligonucleotide sequence containing 0 to 10 modified nucleotides independently; each n p 'and n q ' independently represents an overhanging nucleotide; Here N b 'and Y' do not have the same modifier; and X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It may also be represented by [this method].

[0245] In one embodiment, N a ’ and / or N b ’ includes modification of an alternating pattern.

[0246] 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 from the 5’ end. Preferably, the Y’Y’Y’ motif occurs at positions 11, 12, 13.

[0247] In one embodiment, the Y’Y’Y’ motif consists of all 2’-OMe modified nucleotides.

[0248] 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.

[0249] Thus, the antisense strand can be represented by 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.

[0250] 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.

[0251] 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.

[0252] 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<00​​​​​​​​​​​​​​​​​​​​​​​​

[0255] Each of X', Y', and Z' may be the same as or different from one another.

[0256] Each nucleotide in the sense and antisense strands may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands may be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0257] In one embodiment, the sense strand of the RNAi agent may include a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21nt, where the count starts with the first nucleotide from the 5' end, or optionally, the count starts with the first paired nucleotide inside the double-stranded region from the 5' end, and Y represents a 2'-F modification.

[0258] In one embodiment, the antisense strand may include a Y'Y'Y' motif occurring at positions 11, 12, and 13 of the strand, where the count starts from the first nucleotide from 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.

[0259] Each sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0260] Therefore, the RNAi agent used in the method of the present invention may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the RNAi double strand is given by formula (III): Sense: 5'np -N a -(XXX)iN b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 modified nucleotides independently, with each sequence containing at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0 to 10 modified nucleotides independently; Here each n p ',n p , n q ', and n q Each may or may not be present, and independently represents an overhang nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It is represented by [this].

[0261] 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.

[0262] An example combination of sense and antisense strands that form an RNAi double helix is ​​given 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'np '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5' (IIId) 5'-N a -YYY-N b -3' 3'n p '-N a '-Y'Y'Y'-N b '5' (IIIe) Includes.

[0263] When an RNAi agent is represented by formula (IIIa), each N a This independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0264] When an RNAi agent is represented by formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a This independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0265] When an RNAi agent is represented by formula (IIIc), each N b , N b ' represents an oligonucleotide sequence containing independently modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a This independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0266] When an RNAi agent is represented by formula (IIId), each N b , N b ' represents an oligonucleotide sequence containing independently modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each Na , N a ' represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides independently. a , N a ', N b and N b Each of these independently includes alternating modification patterns.

[0267] When an 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 that are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides.

[0268] In equations (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), each of X, Y, and Z may be the same as or different from one another.

[0269] When an RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), (IIId), and (IIIe), at least one Y nucleotide may form a base pair with one of the Y' nucleotides; or at least two Y nucleotides may form base pairs with the corresponding Y' nucleotides; or all three Y nucleotides may form base pairs with the corresponding Y' nucleotides.

[0270] When an RNAi agent is represented by formula (IIIb) or (IIId), at least one Z nucleotide may form a base pair with one of the Z' nucleotides; or at least two Z nucleotides may form base pairs with the corresponding Z' nucleotides; or all three Z nucleotides may form base pairs with the corresponding Z' nucleotides.

[0271] When an 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; or at least two of the X nucleotides may form a base pair with the corresponding X' nucleotide; or all three of the X nucleotides may form a base pair with the corresponding X' nucleotide.

[0272] In one embodiment, modifications on the Y nucleotide differ from modifications on the Y' nucleotide, modifications on the Z nucleotide differ from modifications on the Z' nucleotide, and / or modifications on the X nucleotide differ from modifications on the X' nucleotide.

[0273] 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 (see 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 The nucleotide 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.

[0274] 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 The nucleotide 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.

[0275] 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 their 5' and 3' ends and optionally bound to a ligand. Each of the activators may target the same gene or two different genes, or each of the activators may target the same gene at two different target sites.

[0276] Various publications describe multimeric RNAi agents that can be used in the methods of the present invention. Such publications include International Publication No. 2007 / 091269, U.S. Patent No. 7858769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887, and International Publication No. 2011 / 031520 (the entire contents of each of these are incorporated herein by reference).

[0277] As will be further detailed below, RNAi agents comprising the binding of one or more carbohydrate moieties to the RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be bound to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent may be substituted with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is bound. Ribonucleotide subunits in which the ribose sugar of the subunit is thus substituted are referred to herein as ribose-substituted modified subunits (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, and sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, such as a fusion ring. The cyclic carrier may be a fully saturated ring system, or may contain one or more double bonds.

[0278] The ligand may be attached to the polynucleotide by a carrier. The carrier comprises (i) at least one “backbone attachment point,” preferably two “backbone attachment points,” and (ii) at least one “tethering attachment point.” When used herein, “backbone attachment point” refers to a functional group, e.g., a hydroxyl group, or generally, a bond that is available and suitable for incorporating the carrier into a ribonucleic acid backbone, e.g., a phosphate backbone, or e.g., a sulfur-containing modified phosphate backbone. “Tethering attachment point” (TAP) refers, in some embodiments, to a constituent ring atom of a cyclic carrier that connects a selected moiety, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point). The moiety may be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Therefore, cyclic supports often contain functional groups, such as amino groups, or generally allow for bonding suitable for incorporation or tethering to the constituent ring of another chemical entity, such as a ligand.

[0279] The RNAi agent may be bound to the ligand via a carrier, which may be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a selinol skeleton or a diethanolamine skeleton.

[0280] In certain specific embodiments, for example, the RNAi agent used in the method of the present invention is an active substance selected from the group of active substances listed in Table 1. These active substances may further contain ligands.

[0281] In one embodiment, the antisense strand of the RNAi agent is 5'-usCfsuugguuacaugAfaaucccasusc-3'(SEQ ID NO: 6), 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3'(Sequence ID 7), 5'-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 8), and The sequence includes a nucleotide sequence selected from the group consisting of 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'-fluoroA, C, G, or U; and s is a phosphorothioate bond; and VP is a 5'-phosphate mimetic).

[0282] In one embodiment, the sense and antisense strands of the RNAi agent are 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugguuacaugAfaaucccasusc-3'(Sequence ID 6); 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3'(Sequence ID 7); 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3'(sequence number 8); and 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and The sequence includes a nucleotide sequence selected from the group consisting of 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'-fluoroA, C, G, or U; and s is a phosphorothioate bond; and VP is a 5'-phosphate mimetic). In another embodiment, the sense and antisense strands comprise the nucleotide sequences 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 10) and 5'-usCfsuugGfuuAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7) (wherein a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate bond). In yet another embodiment, the sense and antisense strands comprise the nucleotide sequences 5'-usgsggauUfuCfAfUfguaaccaagaL96-3' (SEQ ID NO: 15) 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'-fluoroA, C, G, or U; and s is a phosphorothioate bond). In yet another embodiment, the RNAi agent is AD-65492.

[0283] V. Ligand-coupled iRNA Another modification of the iRNA of the present invention involves chemically linking one or more ligands, moieties, or complexes to the RNA, which enhance the activity, cell distribution, or intracellular uptake of the iRNA. Such parts include lipid portions such as the cholesterol portion (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. NYAcad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770) and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); and thioethers such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO Aliphatic chains such as J,1991,10:1111-1118;Kabanov et al.,FEBS Lett.,1990,259:327-330;Svinarchuk et al.,Biochimie,1993,75:49-54); phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654;Shea et al.,Nucl.Acids Res.,1990,18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969-973; or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys.Examples include, but are not limited to, octadecylamine or the hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0284] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides improved affinity to selected targets, such as molecules, cells or cell types, compartments such as intracellular or organelle compartments, tissues or organs or regions of the body, compared to chemical species in which such ligand is absent. The preferred ligand does not participate in double-strand pairing in the double-stranded nucleic acid.

[0285] Ligands 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. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or α-helical peptides.

[0286] The ligand may also include a targeting group such as an antibody that binds to a specific cell type, such as kidney cells, or a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein. The targeting group may be thyroid-stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyhydric lactose, monohydric galactose, N-acetyl-galactosamine, N-acetylglucosamine (gulucoseamine), polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand includes monohydric or polyhydric galactose. In certain embodiments, the ligand includes cholesterol.

[0287] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 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) lithoglycerol Examples include lic acid, O3-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine) and peptide complexes (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole complexes, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, or AP.

[0288] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for a co-ligand; or antibodies, such as antibodies that bind to a specified cell type, such as liver cells. Ligands may also include hormones and hormone receptors. They may also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide chemical species such as polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, or polyhydric fucose. Ligands may be lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators, for example.

[0289] Ligands can be substances such as drugs that can increase the uptake of iRNA agents into cells by disrupting, for example, the cellular microtubules, microfibrils, and / or intermediate filaments, or by disrupting the cellular cytoskeleton. Drugs may include, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0290] In some embodiments, the ligands attached to iRNAs described herein refer to pharmacokinetic modifiers (PK modifiers). Examples of PK modifiers include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, and vitamins. Exemplary PK modifiers include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate bonds are also known to bind to serum proteins, and therefore, for example, short-chain oligonucleotides such as approximately 5-base, 10-base, 15-base, or 20-base oligonucleotides containing multiple phosphorothioate bonds in the main chain are also suitable as ligands (e.g., as PK modulating ligands) in the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0291] The ligand-conjugated oligonucleotides of the present invention may be synthesized using oligonucleotides having pendant-reactive functional groups, such as those derived from the addition of a binding molecule onto the oligonucleotide (described below). These reactive oligonucleotides may be reacted directly with commercially available ligands, synthesized ligands having any of the various protecting groups, or ligands having a binding site to which attachment is possible.

[0292] The oligonucleotides used in the complexes of the present invention may, conveniently and conventionally, be produced through well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including Applied Biosystems (Foster City, Calif.). Alternatively, any other means known in the art for such synthesis may be used. It is also known that other oligonucleotides, such as phosphorothioates and alkylated derivatives, can be prepared using similar techniques.

[0293] In the ligand-conjugated oligonucleotides and sequence-specific binding nucleosides containing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotides or nucleoside precursors, nucleotides or nucleoside complex precursors already containing binding sites, ligand-nucleotide or nucleoside complex precursors already containing ligand molecules, or basic units containing non-nucleoside ligands.

[0294] When using a nucleotide complex precursor that already has a binding site, the synthesis of a sequence-specific bound nucleoside is typically completed, and then the ligand molecule reacts with the binding site to produce a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or bound nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside complexes, in addition to standard and non-standard phosphoramidites that are commercially available and conventionally used in oligonucleotide synthesis.

[0295] A. Lipid complexes In one embodiment, the ligand or complex is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the complex to target tissues, such as non-renal target tissues of the body. Target tissues, for example, could be the liver, including hepatic parenchymal cells. Other molecules capable of binding to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the degradation resistance of the complex, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate the binding of serum proteins, such as HSA.

[0296] Lipid-based ligands can be used for inhibition, for example, by controlling the binding of the complex to target tissues. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the complex to the kidneys.

[0297] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the complex is distributed to non-renal tissues. However, the affinity is preferably not so strong as to prevent the HSA ligand binding from being reversed.

[0298] In another preferred embodiment, the lipid-based ligand binds weakly to or does not bind at all to the HSA so that the complex is preferably distributed to the kidney. Other portions that target renal cells may also be used instead of, or in addition to, the lipid-based ligand.

[0299] In another embodiment, ligands are portions of vitamins, for example, that are taken up by target cells such as proliferating cells. These are particularly useful in 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, and pyridoxal, or other vitamins or nutrients that are taken up by target cells such as liver cells. HSA and low-density lipoprotein (LDL) are also examples.

[0300] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, the cell permeabilizer is amphiphilic. Exemplary cell permeabilizers are peptides such as tat or antennopedia. If the cell permeabilizer is a peptide, it may be modified, including peptidyl mimetic, inverted isomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having lipophilic and oleophobic phases.

[0301] The ligand may be a peptide or a peptide mimetic. Peptidimides (also referred to herein as oligopeptide mimes) are molecules that can fold into a defined three-dimensional structure similar to natural peptides. The addition of peptides and peptide mimes to iRNA agents may affect the pharmacokinetic distribution of the iRNA, such as by enhancing cell recognition and absorption. The peptide or peptide mimetic moiety may be approximately 5 to 50 amino acid lengths, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths.

[0302] Peptides or peptide mimetic drugs may be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, a bound peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane transition sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) may also be target moieties. The peptide moiety may be a “delivery” peptide capable of transporting a number of polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. Sequences from, for example, HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) and Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 14)) have been shown to function as delivery peptides. Peptides or peptide mimetic drugs can be encoded by random sequences of DNA, such as peptides identified from phage-display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). For cell targeting purposes, examples of peptides or peptide mimetic drugs anchored to dsRNA activators via incorporated monomer units include arginine-glycine-aspartate (RGD) peptides or RGD mimetic drugs. The peptide moiety can range in length from approximately 5 to 40 amino acids. The peptide moiety may have structural modifications that increase stability or induce conformational properties. Any of the structural modifications described below may be used.

[0303] 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 specific tissues. Examples of RGD-containing peptides and peptide mimetic agents include D-amino acids and synthetic RGD mimetic agents. In addition to RGD, other moieties that target integrin ligands may be used. Preferred ligand complexes target PECAM-1 or VEGF.

[0304] "Cell-permeable peptides" can penetrate cells such as microbial cells, including bacterial or fungal cells, or mammalian cells, including human cells. Microbial cell-permeable peptides may be, for example, α-helical linear peptides (e.g., LL-37 or ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphimotic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0305] C. Carbohydrate complex In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, “carbohydrate” means a carbohydrate itself, which consists of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound which has as part a carbohydrate portion consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Examples of specific monosaccharides include sugars with a TTR or higher (e.g., TTR, C6, C7, or C8); disaccharides include sugars having two or three monosaccharide units (e.g., TTR, C6, C7, or C8).

[0306] In one embodiment, the carbohydrate complex used in the composition and method of the present invention is a monosaccharide. In another embodiment, the carbohydrate complex used in the composition and method of the present invention is [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0307] In one embodiment, the monosaccharide is N-acetylgalactosamine, for example, [ka] That is the case.

[0308] Other representative carbohydrate complexes used in the embodiments described herein include, but are not limited to, [ka] (Formula XXIII) (where one of X or Y is an oligonucleotide and the other is hydrogen) is included.

[0309] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is conjugated to the iRNA agent of the present invention via a trivalent linker.

[0310] In one embodiment, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative conjugated to an iRNA agent. In another embodiment, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently conjugated to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0311] In some embodiments, for example, when the two strands of the iRNA agent of the present invention are part of one larger molecule linked by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently contain GalNAc or a GalNAc derivative linked via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the two strands.

[0312] In some embodiments, the carbohydrate complex further comprises one or more further ligands, such as, but not limited to, PK modifiers and / or cell-penetrating peptides.

[0313] Further carbohydrate complexes suitable for use in the present invention include those described in the PCT Publications International Publication No. 2014 / 179620 and International Publication No. 2014 / 179627 (the entire contents of each of these publications are incorporated herein by reference).

[0314] D. Linker In some embodiments, the complexes or ligands described herein may be attached to the iRNA oligonucleotide by various linkers, which may be cleavable or incleavable.

[0315] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, such as by covalently bonding two parts of the compound together. Linkers are typically directly bonded, or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryl, heteroaryl, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls, This includes, but is not limited to, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkylherocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynylherocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenyl heteroaryl, alkynylhereroaryl, and other atomic chains, one or more of which 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 The linker can be interrupted or terminated by hydrogen, acyl, aliphatic, or substituted aliphatic atoms. In one embodiment, the linker is approximately 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.

[0316] The cleavable linking group is sufficiently stable outside the cell but is cleaved upon entry into the target cell, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more, or at least about 100 times faster in the target cell or under a first standard condition (which may be selected to mimic or be equivalent to intracellular conditions) than in the target blood or under a second standard condition (which may be selected to mimic or be equivalent to conditions found in blood or serum).

[0317] Cleavable linkers are susceptible to the influence of cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common in cells than in serum or blood, or are found at higher levels or activity. Examples of such degradable agents include oxidative or reductases or reducing agents such as mercaptans present in cells that can degrade redox-cleavable linkers by reduction, and redox-selected or non-substrate-specific redox agents selected for specific substrates; esterases; agents that can create acidic environments, such as endosomes or those that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0318] Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. While human serum has a pH of 7.4, the mean intracellular pH is slightly lower, ranging from approximately 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 approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a favorable pH, thereby releasing cationic lipids from ligands within the cell or to desired compartments of the cell.

[0319] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into a linker may depend on the target cell. For example, a ligand targeting the liver may link to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and therefore linkers are cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0320] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synovial cells.

[0321] Generally, the suitability of candidate cleavable linkers can be evaluated by testing the ability of a degrading agent (condition) to cleave the candidate linker. It is also desirable to test the candidate cleavable linker's resistance to cleavage in the blood or in contact with other non-target tissues. Therefore, the relative susceptibility to cleavage between a first and second condition can be determined, with the first condition selected to demonstrate cleavage in target cells and the second condition selected to demonstrate cleavage in other tissues or in biological fluids such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or in whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and then confirm it with further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0322] i. Redox-cleavable linking groups 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 (-SS-). Methods described herein can be relied upon to determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent. For example, a candidate may be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. One candidate compound is cleaved by up to about 10% in blood. In other embodiments, useful candidate compounds are degraded at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using a standard enzyme kinetics assay under conditions selected to mimic an extracellular medium, compared to conditions selected to mimic an extracellular medium.

[0323] ii. Phosphate-based cleavable linking groups 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 degrades or hydrolyzes the phosphate group. An example of an agent that cleaves the phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0324] iii. Acid-cleavable linking group In another embodiment, the cleavable linker includes 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 with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, or less) or by an active agent such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles such as endosomes and lysosomes can provide an environment for cleaving acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable linking group may 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 (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 methods similar to those described above.

[0325] iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. The ester-based cleavable linking group is cleaved in the cell by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkylylene groups. The ester-based cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0326] v. Peptide-based cleavage groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved in cells by enzymes such as peptidases and proteases. The peptide-based cleavable linking group is a peptide bond, which is formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable linking group does not contain an amide group (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond that is formed between amino acids to produce peptides and proteins. The peptide-based cleavable linking group is generally limited to peptide bonds (i.e., amide bonds) that are formed between amino acids to produce peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linking group 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.

[0327] In one embodiment, the iRNA of the present invention is coupled to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrates coupled to the linker of the composition and method of the present invention include: [ka] [ka] (In the formula, Examples include, but are not limited to, a (where either X or Y is an oligonucleotide and the other is hydrogen) array.

[0328] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker.

[0329] In one embodiment, the dsRNA of the present invention is Formulas (XXXII) to (XXXV), [ka] (In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent each occurrence from 0 to 20, and the repeating units may be identical or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C Each occurrence is independently of the others: absence, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each occurrence is independently of the others: absent, alkylene, substituted alkylene, and one or more methylene groups: O, S, S(O), SO2, N(R) N ), C(R')=C(R''), C≡C or C(O) may be interrupted or terminated by one or more of these; R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B, R 5C Each occurrence is independently: absence, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocycline; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, each occurrence is independently a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a It is conjugated with a divalent or trivalent branched linker, selected from a group of structures represented by either H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are Formula (XXXVI), [ka] (In the formula, L 5A , L 5B and L 5C It is particularly useful when used in conjunction with RNAi agents to inhibit the expression of target genes (such as monosaccharides represented by GalNAc derivatives).

[0330] 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.

[0331] Representative U.S. patents teaching the preparation of RNA complexes are, as are incorporated herein by reference in their entirety: 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; and U.S. Patent No. 5,591,584. Detailed Statement; 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 Details Document; 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 Nos. 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646 and 8,106,022 are examples, but are not limited to these.

[0332] It is not necessary for all positions in a given compound to be uniformly modified; in fact, two or more of the aforementioned modifications can be incorporated into a single compound, or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0333] In the context of this invention, a "chimeric" iRNA compound or "chimeras" is an iRNA compound, preferably a dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to give the iRNA increased resistance to nuclease degradation, increased intracellular uptake, and / or increased binding affinity to a target nucleic acid. The additional region of the iRNA may act as an enzyme substrate capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded molecule. Therefore, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region, comparable results are often obtained with shorter iRNAs. Cleavage of RNA targets can conventionally be detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0334] In some cases, the RNA of iRNA can be modified with non-ligand groups. Several non-ligand molecules are conjugated to iRNA to enhance its activity, cell distribution, or intracellular uptake, and procedures for performing such conjugations are available in the academic literature.These non-ligand portions include lipid portions 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. NYAcad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids 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 & It contains Nucleotides (1995, 14:969), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents teaching the preparation of such RNA complexes are listed above. A typical conjugation protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group is then reacted with a molecule that conjugates the amino group using an appropriate coupling or activating reagent. The conjugation reaction can be carried out in the solution phase while the RNA is still bound to a solid support, or following RNA cleavage. Purification of the RNA complex by HPLC typically yields a pure complex.

[0335] VI. Delivery of iRNA according to the present invention The delivery of the iRNA of the present invention to cells, for example, to cells within a human subject (e.g., a subject requiring it, e.g., a subject with a disease, disorder, or condition related to contact activation pathway gene expression), can be achieved in several different ways. For example, delivery may be carried out by contacting cells with the iRNA of the present invention, either in vitro or in vivo. In vivo delivery may also be carried out directly by administering a composition containing the iRNA, such as dsRNA, to the subject. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode and induce the expression of the iRNA. These alternatives are discussed further below.

[0336] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNA of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, whose entire contents are incorporated herein by reference). For in vivo delivery, factors to be considered for delivering the iRNA molecule include, for example, the biological stability of the delivery molecule, prevention of nonspecific effects, and accumulation of the delivery molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or transplantation into tissue or local administration of the formulation. Local administration to the treatment site maximizes the local concentration of the active substance, limits exposure of systemic tissues to the active substance which could otherwise be harmed or degraded by the active substance, and allows for administration of lower total doses of the iRNA molecule. Several studies have shown successful gene product knockdown when iRNA is administered locally. For example, intravitreal injection of VEGF dsRNA 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 demonstrated the prevention of neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol.Ther.11:267-274) and extended the survival time of mice with tumors (Kim, WJ., et al (2006) Mol.Ther.14:343-350; Li, S., et al (2007) Mol.Ther.15:515-523).RNA interference can be administered to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602), and to the lungs by intranasal administration (Howard, KA., 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) Successful local delivery has been demonstrated. To treat diseases, or to administer iRNA systemically, RNA can be modified or, alternatively, delivered using drug delivery systems; both methods act to prevent rapid degradation of dsRNA by endogenous and exonucleases. Modification of RNA or pharmaceutical carriers can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical bonding of lipophilic groups such as cholesterol to enhance intracellular uptake and prevent degradation. For example, when an iRNA that counteracts ApoB, which is conjugated to the lipophilic cholesterol portion, was systemically injected into mice, apoB mRNA knockdown was induced in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). The conjugation of iRNA to aptamers has been shown to suppress tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNA may be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of the iRNA molecule (which is negatively charged) and also enhance interactions with the negatively charged cell membrane, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may be induced to bind to iRNA or form vesicles or micelles that enclose the iRNA (see, e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of iRNA when administered systemically. Methods for preparing 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, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), cited above; Verma, UN., et al (2003), cited above), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441:111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME., et al. Examples include al (2008) Pharm. Res., published online on August 16; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D., 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. Methods of administering iRNA and cyclodextrin and pharmaceutical compositions are described in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0337] A. The iRNA of the present invention encoded by the vector iRNAs targeting contact activation pathway genes can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International Publication No. 00 / 22113 of the International PCT Publication, Conrad, International Publication No. 00 / 22114 of the International PCT Publication, and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a few hours to several weeks) or persistent (from several weeks to several months or more), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which may be embedded or non-embedded vectors. The transgene can also be constructed to allow it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0338] Individual iRNA strands or strand groups can be transcribed from a promoter on an expression vector. When expressing two separate strands to generate, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (e.g., by transfusion or infection). Alternatively, the individual strands of the dsRNA can be transcribed by promoters 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.

[0339] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for iRNA expression described herein can be generated using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial suppliers. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors may include systemic administration, such as intravenous or intramuscular administration; administration to target cells explanted from a patient and subsequent reintroduction into the patient; or any other means that allows introduction into desired target cells.

[0340] iRNA expression plasmids can be translocated into target cells as complexes with cationic lipid carriers (e.g., oligofectamines) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid translocation for iRNA-mediated knockdown, targeting different regions of the target RNA over a period of more than one week, is also explored in this invention. Successful introduction of the vector into host cells can be monitored using various known methods. For example, transient translocation can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable translocation into cells in vitro can be ensured by using markers that provide the translocated cells with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0341] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentivirus vectors and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, including vaccinia virus vectors, or avipox, including canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may optionally include a viral sequence for translocation. Alternatively, the construct may be incorporated into an episomal replication vector, such as EPV and EBV vectors. Constructs for the recombinant expression of iRNAs generally require regulatory factors, such as promoters and enhancers, to ensure iRNA expression in target cells. Other aspects of vectors and constructs that are considered are described in more detail below.

[0342] A vector useful for delivering iRNA contains sufficient regulatory factors (promoters, enhancers, etc.) to express the iRNA in the desired target cells or tissues. These regulatory factors can be selected to provide either constitutive or regulatory / inducible expression.

[0343] iRNA expression can be precisely regulated using inductive regulatory sequences sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J.8:20-24). Suitable inductive expression systems for regulating dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, dimerizing chemical inducers, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0344] 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 the correct packaging of the viral genome and its integration into host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors to facilitate delivery of the nucleic acid to the patient. For more details on retroviral vectors, see Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to generate stem cells that exhibit higher resistance to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors that may be considered include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; U.S. Patent No. 5,665,557; and U.S. Patent No. 5,981,276, which are incorporated herein by reference.

[0345] Adenoviruses are also being considered for use in iRNA delivery according to the present invention. Adenoviruses are particularly attractive vehicles for delivering genes, for example, to the airway epithelium. Adenoviruses infect the airway epithelium naturally, causing mild disease. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapies. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors for transferring genes into the airway epithelium of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); International Publication No. 94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing the iRNA addressed in this invention, a method for constructing a recombinant AV vector, and a method for delivering the vector to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0346] 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 may be expressed as two distinct complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNAs addressed in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described herein by reference in their entirety: Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Publication No. 94 / 13788; and International Publication No. 93 / 24641.

[0347] Other viral vectors suitable for delivering the iRNA of the present invention include, for example, vaccinia viruses such as modified virus Ankara (MVA) or attenuated vaccinia such as NYVAC, and poxviruses such as avipox such as fowlpox or canarypox.

[0348] The affinity of a viral vector can be modified, if necessary, by pseudotyping the vector with coat proteins or other surface antigens from other viruses, or by substitution with capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mocola, etc. AAV vectors can be genetically engineered to target different cells by expressing different capsid protein serotypes; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, whose entire disclosure is incorporated herein by reference.

[0349] A vector-based drug may contain a vector in an acceptable diluent, or a sustained-release matrix in which a gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be generated intact from recombinant cells, the drug may contain one or more cells that generate the gene delivery system.

[0350] VII. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA described herein for use in the methods of the present invention. In one embodiment, the present invention provides a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier. The iRNA-containing pharmaceutical composition is useful for treating diseases or disorders related to the expression or activity of TTR genes. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery, for example, by subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma, for example, 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 TTR genes. In one embodiment, the iRNA agent of the present invention, for example, a dsRNA agent, is formulated for subcutaneous administration in a pharmaceutically acceptable carrier.

[0351] The pharmaceutical composition may be administered by intravenous infusion over a period of time, for example, over periods of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or approximately 25 minutes. Administration may be repeated for one month, two months, three months, four months, or longer, for example, regularly, for example, weekly, every other week (i.e., every two weeks). Administration may also be repeated, for example, on a monthly or quarterly basis, for example, every 12 weeks. After the initial treatment plan, the therapeutic agent may be administered on a lower frequency basis. For example, after weekly or every other week for three months, administration may be repeated once a month for six months or more.

[0352] The pharmaceutical composition may be administered once daily, or the iRNA may be administered in two or three or more partial doses at appropriate intervals throughout the day, or even by delivery via continuous infusion or controlled-release formulation. In this case, the amount of iRNA contained in each partial dose must be correspondingly smaller in order to achieve the total daily dose. The dosing unit may also be formulated for delivery over several days using conventional sustained-release formulations that provide sustained release of iRNA over several days, for example. Sustained-release formulations are well known in the art and are particularly useful for the delivery of the active ingredient to a specific site, which may be used with the active ingredient of the present invention. In this embodiment, the dosing unit contains a number of corresponding daily doses.

[0353] In another embodiment, a single dose of the pharmaceutical composition may be administered over a longer period, with subsequent doses given 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 dose of the pharmaceutical composition of the present invention is administered once a week. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered twice a month. In yet another embodiment, a single dose of the pharmaceutical composition of the present invention is administered monthly. In yet another embodiment, a single dose of the pharmaceutical composition of the present invention is administered quarterly. In yet another embodiment, the pharmaceutical composition of the present invention is administered once every 4 months in a single dose. In yet another embodiment, the pharmaceutical composition of the present invention is administered once every 5 months in a single dose. In yet another embodiment, the pharmaceutical composition of the present invention is administered once every 6 months in a single dose.

[0354] Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective dose of the composition may consist of a single treatment or a series of treatments. The effective doses and in vivo half-lives of individual iRNAs incorporated in this invention may be estimated using conventional procedures or based on in vivo studies using appropriate animal models, as described elsewhere in this specification.

[0355] The pharmaceutical compositions of the present invention may be administered in several ways, depending on whether topical or systemic treatment is desired and on the treatment area. Administration may be topical (e.g., by a transdermal patch), transpulmonary by inhalation or blowing of powder or fume, including by a nebulizer; intratracheal, intranasal, transepidermal and transdermal, oral or parenteral. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal administration via an implantable device, for example; or intracranial administration, such as within the brain parenchyma, subarachnoid space or ventricles.

[0356] iRNAs can be delivered in a manner that targets specific tissues, such as the liver (e.g., parenchymal cells of the liver).

[0357] 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. Covered condoms, gloves, etc., may also be useful. Suitable topical formulations include those in which the iRNA addressed in this invention is in a miscible material with topically delivered substances such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNAs discussed in this invention can be encapsulated within liposomes, or can form complexes with them, particularly with cationic liposomes. Alternatively, the iRNAs can form complexes 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-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Examples include, but are not limited to, alkyl esters (e.g., isopropylmyristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0358] A. iRNA preparations containing membrane-like molecular assemblies The iRNAs used in the compositions and methods of the present invention may be formulated for delivery within membrane-like molecular assemblies, such as liposomes or micelles. In this specification, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one or more bilayers. Liposomes include monolayer or multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition but may optionally. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to a biological membrane, when liposomes are applied to tissue, the liposomal bilayer fuses with the cell membrane bilayer. As the fusion of the liposome and cell progresses, the internal aqueous contents containing the iRNA are delivered into the cell, where the iRNA can specifically bind to target RNA and mediate its delivery. In some cases, liposomes are also specifically targeted, for example, to induce iRNAs into specific cell types.

[0359] Liposomes containing iRNA agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed without the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid complex. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid, and lauroyl sarcosine. Next, the iRNA agent preparation is 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 the iRNA agent liposome preparation.

[0360] If necessary, a support compound, for example, that aids condensation may be added during the condensation reaction by controlled addition. For example, the support compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). pH may also be adjusted to aid condensation.

[0361] A method for generating a stable polynucleotide delivery vehicle by incorporating a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle is further described, for example, in International Publication No. 96 / 37194, which is incorporated herein by reference in its entirety. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. al.Biochim.Biophys.Acta 557:9,1979;Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978;Mayhew,et al.Biochim.Biophys.Acta 775:169,1984;Kim,et al.Biochim.Biophys.Acta 728:339,1983; and Fukunaga, et al. This may also include one or more embodiments of the exemplary methods described in al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing appropriately sized lipid aggregates for use as delivery vehicles include sonication and combinations of freeze-thaw and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). If consistently small (50–200 nm) and relatively uniform aggregates are desired, micro-solution preparation may be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable to packing iRNA drug preparations into liposomes.

[0362] Liposomes are broadly classified into two 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 into the endosome. Due to the acidic pH inside the endosome, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0363] pH-sensitive or negatively charged liposomes do not form complexes with nucleic acids; rather, they encapsulate them. Because nucleic acids and lipids both have similar charges, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used in culture to deliver nucleic acids encoding thymidine kinase genes to cell monolayers. Expression of exogenous genes was detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0364] One major type of liposome composition contains phospholipids in addition to naturally derived phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusion liposomes are mainly formed from dioleoyl sphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC. Yet another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0365] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; U.S. Patent Nos. 5,171,678; International Publication No. 94 / 00569; International Publication No. 93 / 24640; International Publication No. 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.

[0366] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied, and their efficacy in drug delivery to the skin has been evaluated. Cyclosporine A was delivered into the dermis of mouse skin using nonionic liposome formulations containing Novasome® I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome® II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results suggested that such nonionic liposome systems are effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. STPPharma.Sci., 1994, 4, 6, 466).

[0367] Liposomes also include “stereostabilized” liposomes, which, as used herein, refer to liposomes containing one or more specialized lipids, which, when incorporated into liposomes, result in an improved cyclic lifespan compared to liposomes lacking such specialized lipids. An example of a stereostabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome contains (A) monosialoganglioside G M1(B) These contain one or more glycolipids, or are derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While we do not wish to be constrained by any particular theory, in the art, in sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivativeized lipids, the improvement in the circulating half-life of these sterically stabilized liposomes is thought to be due to reduced uptake by reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0368] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NYAcad. Sci., 1987, 507, 64) described monosialoganglioside G M1 The ability of galactocerebroside sulfate and phosphatidylinositol to improve the half-life of liposomes in the blood has been reported. These findings were described in detail by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Both U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924, both granted to Allen et al., describe (1) sphingomyelin and (2) ganglioside G M1 Liposomes comprising or galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0369] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot efficiently fuse with the plasma membrane, but they can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0370] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; they can encapsulate a wide range of water- and lipid-soluble drugs; and they can protect iRNA agents encapsulated within their internal compartments from metabolism and degradation (Rosoff, “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Key considerations in the preparation of liposomal formulations include the lipid surface charge, vesicle size, and aqueous capacity of the liposomes.

[0371] Using the positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), small liposomes can be formed, which spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with negatively charged lipids in the cell membrane of tissue culture cells, resulting in iRNA delivery (for a description of DOTMA and its use in combination with DNA, see, for example, Felgner, Plet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355).

[0372] The ADOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin® (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to cultured tissue cells, and it contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complex thus prepared spontaneously adheres to negatively charged cell surfaces, 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-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl portion is linked by an ester rather than an ether linkage.

[0373] Other reported cationic lipid compounds include those conjugated to a variety of moieties, such as carboxyspermine conjugated to one of two lipid types, and compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam®, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES") (see, for example, U.S. Patent No. 5,171,678).

[0374] Another cationic lipid complex involves lipid derivatization by cholesterol ("DC-Chol") combined with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for translocation 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 translocation 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 oligonucleotide delivery are described in International Publication No. 98 / 39359 and International Publication No. 96 / 37194.

[0375] Liposome formulations are particularly well-suited for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver 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 tissues, such as within the skin. For example, liposomes can be applied topically. Topical delivery of therapeutic drugs formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol.2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. Jand 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. Rand Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987.

[0376] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied, and their efficacy in drug delivery to the skin has been determined. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0377] iRNA-containing liposomes can be highly deformable. Such deformability allows the liposomes to penetrate pores smaller than the average radius of the liposome. Transferosomes, for example, are a type of deformable liposome. Transferosomes can be created by adding surface edge activators, usually surfactants, to a standard liposome composition. Transferosomes containing iRNA agents can be delivered subcutaneously, for example by infection, to deliver the iRNA agent to keratinocytes in the skin. To cross intact mammalian skin, the lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of a suitable transcutaneous gradient. Furthermore, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and often self-load.

[0378] Other formulations relating to the present invention are described in the PCT International Publication No. 2008 / 042973 (the entire contents of which are incorporated herein by reference).

[0379] Transfersomes are yet another type of liposome, highly deformable lipid aggregates, that are attractive candidates for drug delivery vehicles. Because they are so highly deformable, transfersomes can be described as lipid droplets that can easily penetrate through pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to skin pore shapes), self-repair, and often reach and self-load their targets without fragmentation. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0380] 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 the many different types of surfactants, both natural and synthetic, is the use of the hydrophile / lipophile balance (HLB). The properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0381] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used across a wide pH range. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Examples of nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, also belong to this class. Polyoxyethylene surfactants are the most commonly found components of the nonionic surfactant class.

[0382] Surfactants are classified as anionic when their molecules retain a negative charge when dissolved or dispersed in water. Examples of anionic surfactants include carboxylates such as soap, acyl lactylate, acylamides of amino acids, sulfate esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0383] Surfactants are classified as cationic if their molecules retain a positive charge when dissolved or dispersed in water. Examples of cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used components in this class.

[0384] A surfactant is classified as amphoteric if its molecule has the ability to possess either a positive or negative charge. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phospholipids.

[0385] The use of surfactants in pharmaceuticals, formulations, and emulsions is outlined (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0386] The iRNA used in the method of the present invention may also be provided as a micelle formulation. A “micelle” is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. The opposite configuration exists if the environment is hydrophobic.

[0387] Mixed micelle formulations suitable for transdermal delivery include siRNA compositions and alkali metals C8-C8. 22The mixture may be prepared by mixing an aqueous solution of an alkyl sulfate and a micelle-forming compound. Examples of 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, borage oil, evening primrose oil, menthol, trihydroxyoxocolanyglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogues, polydocanol alkyl ether and its analogues, 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 will form regardless of how the components are mixed substantially, but vigorous mixing is preferable to provide smaller micelles.

[0388] In one method, a first micelle composition is prepared containing an siRNA composition and at least an alkali metal alkyl sulfate. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, 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.

[0389] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the preparation and protect it from 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 mixed micelle composition has been formed.

[0390] To deliver a micelle formulation as a spray, the formulation can be placed in a fumigant metering and dispensing device, and the spray can be loaded into the device. Under pressurization, the spray is in liquid form within the metering and dispensing device. The ratio of components is adjusted so that the aqueous phase and the spray phase are one, i.e., a single phase. If there are two phases, the metering and dispensing device needs to be shaken, for example, before dispersing a portion of the contents through a metering valve. The dispensing dose of the pharmaceutical is sprayed in a fine mist from the metering valve.

[0391] Examples of spraying agents include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ethers, and diethyl ethers. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0392] The specific concentration of essential components can be determined by relatively simple experimental methods. For oral absorption, it is often desirable to increase the dose to, for example, at least two or three times the dose administered by injection or via the gastrointestinal tract.

[0393] B. Lipid particles For example, iRNAs such as the dsRNA of the present invention may be formed by completely encapsulating them in a lipid formulation such as LNP or other nucleic acid-lipid particles.

[0394] In the use herein, the term “LNP” refers to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid complexes). LNPs exhibit a long circulatory lifetime following intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic applications. Examples of LNPs include “pSPLP,” which contains encapsulating condensant-nucleic acid complexes as described in International Publication No. 00 / 03683. The particles of the present invention are substantially non-toxic and 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. In addition, when present in the nucleic acid-lipid particles of the present invention, the nucleic acids are resistant to nuclease degradation 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 International Publication No. 96 / 40964.

[0395] In one embodiment, the ratio (mass / mass ratio) of lipids to drugs (e.g., lipid to dsRNA ratio) is in the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1. Ranges between the ranges cited above are also considered to be part of the present invention.

[0396] 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-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylamine. Minopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleyoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleythio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-Linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyoxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (propanedio) (DOAP), 1,2-dilinoleyloxo-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) The analogs thereof may be (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazandiyl)didodecane-2-ol (Tech G1), or mixtures thereof. Cationic lipids can constitute approximately 20 mol% to 50 mol% or 40 mol% of the total lipids present in the particles.

[0397] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0398] In one embodiment, the lipid-siRNA particles consist of 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (molar percentage), with a particle size of 63.0 ± 20 nm and an siRNA / lipid ratio of 0.027.

[0399] Ionic / noncationic lipids include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerin (DOPG), dipalmitoyl phosphatidylglycerin (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine-4-(N-maleimide). These may include, but are not limited to, anionic or neutral lipids, such as 1-Cyl-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. If cholesterol is present, noncationic lipids may constitute about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0400] Conjugated lipids that inhibit particle aggregation may include, without limitation, polyethylene glycol (PEG) lipids, such as PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, and PEG-ceramide (Cer), or mixtures thereof. PEG-DAA complexes may include, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C8). The amount of conjugated lipids that prevent particle aggregation may be 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0401] In some embodiments, the nucleic acid-lipid particles further contain, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particles, which is cholesterol.

[0402] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be prepared using the lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, whose contents are 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. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be combined in a molar ratio, for example, 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (for example, in sodium acetate at pH 5) so 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 during 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 replaced with phosphate-buffered saline (PBS) at approximately pH 7, such as approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. [ka]

[0403] The LNP01 formulation is described, for example, in International Publication No. 2008 / 042973, which is incorporated herein by reference.

[0404] Additional exemplary lipid dsRNA preparations are listed in Table 1.

[0405] [Table 1]

[0406] [Table 2]

[0407] [Table 3]

[0408] Formulations comprising SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. 2009 / 127060, filed on April 15, 2009, which is incorporated herein by reference.

[0409] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366 filed on 29 January 2009; U.S. Provisional Patent Application No. 61 / 156,851 filed on 2 March 2009; U.S. Provisional Patent Application No. [number missing] filed on 10 June 2009; U.S. Provisional Patent Application No. 61 / 228,373 filed on 24 July 2009; U.S. Provisional Patent Application No. 61 / 239,686 filed on 3 September 2009; and International Application PCT / US2010 / 022614 filed on 29 January 2010 (as incorporated herein by reference).

[0410] Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed on June 10, 2010 (the full contents of which are incorporated herein by reference).

[0411] Formulations containing ALNY-100 are described, for example, in International Patent Application PCT / US09 / 63933, filed on November 10, 2009 (as referred herein).

[0412] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed on May 5, 2009, and International Application PCT / US10 / 33777, filed on May 5, 2010 (as referred herein).

[0413] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, suspensions or solutions in water or aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. In some embodiments, the oral formulation is administered in combination with one or more osmotic surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, combinations of osmotic enhancers are used, such as fatty acid / salt combined with bile acid / salt. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA addressed in this invention may be delivered orally in granular 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, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pullulans, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexyl Examples include acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, methyl polyacrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparations 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, respectively, which are incorporated herein by reference.

[0414] Compositions and formulations for parenteral, intracerebral (intracerebral), subarachnoid, intraventricular, or intrahepatic administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including but not limited to osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0415] Examples of the pharmaceutical compositions 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 components, including, but are not limited to, pre-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.

[0416] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage forms, can be prepared according to the prior art well known in the pharmaceutical industry. Such art involves the step of combining the active ingredient with a pharmaceutical carrier or excipient. Generally, formulations are prepared by uniformly and closely combining the active ingredient with a liquid carrier or an ultrafine particle solid carrier or both, and then shaping the product if necessary.

[0417] 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 may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspensions may also contain stabilizers.

[0418] C. Additional formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid, usually in the form of droplets with a diameter greater than 0.1 μm (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p.335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301. Emulsions are often biphasic systems containing two immiscible liquid phases that are closely mixed and dispersed from one another. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed in a bulk oily phase as microdroplets, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed in a bulk aqueous phase as microdroplets, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to a dispersed phase and an active agent, which may exist as a solution in either an aqueous or 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 needed. Pharmaceutical emulsions can also be multi-phase emulsions, such as oil-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer specific advantages that simple two-component emulsions do not. Among these, a multi-phase emulsion in which individual oil droplets of an o / w emulsion surround smaller water droplets constitutes a w / o / w emulsion. Similarly, an oil droplet system encapsulated in small water spheres and stabilized within a continuous oily phase provides an o / w / o emulsion.

[0419] Emulsions are characterized by having little to no thermodynamic stability. Often, the dispersed or discontinuous phases of an emulsion are well dispersed externally or within the continuous phase and maintained in this form through emulsifiers or means of increasing the formulation viscosity. In the case of emulsion-type ointment bases and creams, any of the emulsion phases may be semi-solid or solid. Another means of stabilizing an emulsion involves the use of emulsifiers, which may be incorporated into any of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorbent bases, and finely dispersed solids (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0420] Synthetic surfactants, also known as surfactants, have a wide range of applications in emulsion formulations and are outlined 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, NY, volume 1, p.285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199). Surfactants are typically amphiphilic, containing both hydrophilic and hydrophobic moieties. The ratio of hydrophilic to hydrophobic groups is called the hydrophilic / lipophilic balance (HLB) of a surfactant and is a useful means of classifying and selecting surfactants in the preparation of pharmaceutical formulations. Surfactants can be classified into different classes based on the properties of their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0421] Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorbent bases with hydrophilic properties that can absorb water and form w / o emulsions, such as anhydrous lanolin and hydrophilic petrolatum, still maintain their semi-solid viscosity. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, particularly in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-expanding 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.

[0422] 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, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199).

[0423] Examples of hydrophilic colloids 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., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These disperse in water or swell 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 outer phase.

[0424] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can readily support microbial growth; therefore, preservatives are frequently incorporated into these formulations. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene; reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0425] The application of emulsion formulations via cutaneous, oral, and parenteral routes, and methods for manufacturing them, are outlined in the literature. (See, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are widely used due to their ease of preparation and efficiency in terms of absorption and bioavailability (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199). Mineral oil-based laxatives, fat-soluble vitamins, and high-fat nutritional supplements are among the materials commonly administered orally as o / w emulsions.

[0426] ii. Microemulsion In one embodiment of the present invention, the iRNA and nucleic acid composition is prepared as a microemulsion. A microemulsion can be defined as a single optically isotropic and thermodynamically stable solution of water, oil, and an amphiphilic substance (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, 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, which is generally an alcohol of intermediate chain length, to form a clear system. Therefore, microemulsions are described as thermodynamically stable, isotropically transparent dispersions 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 typically prepared through a combination of 3 to 5 components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used, as well as 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).

[0427] Phenomenological approaches using phase diagrams have been extensively studied, providing those skilled in the art with comprehensive knowledge regarding the formulation 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, NY, volume 1, p.245; Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs into spontaneously formed, thermodynamically stable droplet formulations.

[0428] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with co-surfactants. Typically, co-surfactants, which are short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant coating, resulting in an irregular coating due to gaps between surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may, but is not limited to, water, aqueous solutions of pharmaceuticals, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0429] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as 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 Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when compounding heat-unstable drugs, peptides, or iRNAs. Microemulsions have proven effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve local intracellular uptake of iRNAs and nucleic acids.

[0430] The microemulsion of the present invention may also contain additional components an...

Claims

1. A pharmaceutical composition for treating human subjects suffering from or at risk of developing TTR-related disease, wherein the pharmaceutical composition comprises a double-stranded RNAi agent or a salt thereof administered in a constant dose of about 25 mg to about 50 mg. Here, TTR-related diseases are selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial polyamyloid neuropathy (FAP), familial amyloid cardiomyopathy (FAC), pia mater / central nervous system (CNS) amyloidosis, amyloid vitreous opacity, carpal tunnel syndrome, and hyperthyroxinemia. Here, the double-stranded RNAi agent includes a sense strand complementary to the antisense strand. Here, the sense strand includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10, and the antisense strand includes the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusuc-3' of SEQ ID NO: 7, Herein, a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate bond, a pharmaceutical composition (excluding a pharmaceutical composition in which the double-stranded RNAi agent is administered in a constant dose of about 25 mg).

2. A pharmaceutical composition for improving at least one indicator of neurological impairment or quality of life in human subjects suffering from or at risk of developing TTR-related disease, wherein the pharmaceutical composition comprises a double-stranded RNAi agent or a salt thereof administered in a constant dose of about 25 mg to about 50 mg. Here, TTR-related diseases are selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial polyamyloid neuropathy (FAP), pia mater / central nervous system (CNS) amyloidosis, and carpal tunnel syndrome. Here, the double-stranded RNAi agent includes a sense strand complementary to the antisense strand. Here, the sense strand includes the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10, and the antisense strand includes the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusuc-3' of SEQ ID NO: 7, Herein, a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; and s is a phosphorothioate bond, a pharmaceutical composition (excluding a pharmaceutical composition in which the double-stranded RNAi agent is administered in a constant dose of about 25 mg).

3. The pharmaceutical composition according to claim 2, wherein the aforementioned index is an index of neurological impairment.

4. The pharmaceutical composition according to claim 3, wherein the neurological impairment index is a neurological impairment (NIS) score or a revised NIS (mNIS+7) score.

5. A pharmaceutical composition according to claim 2, wherein the index is a quality of life index, the quality of life index is selected from the group consisting of the SF-36® health survey score, Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, NIS-W score, Rasch-built Overall Disability Scale (R-ODS) score, Composite Autonomic Symptom Score (COMPASS-31), Median Body Mass Index (mBMI) score, 6-Minute Walk Test (6MWT) score, and 10-Minute Walk Test score.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human subject has a TTR gene mutation associated with the manifestation of TTR-related disease.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the pharmaceutical composition reduces amyloid TTR deposition in the human subject.

8. The pharmaceutical composition according to claim 7, wherein the ATTR is hereditary ATTR (h-ATTR).

9. The pharmaceutical composition according to claim 7, wherein the ATTR is a non-hereditary ATTR (wt ATTR).

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the double-stranded RNAi agent or a salt thereof is administered to the human subject by an administration means selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic vessel, cerebrospinal fluid, and any combination thereof.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the double-stranded RNAi agent or a salt thereof is administered to the human subject by subcutaneous, intramuscular, or intravenous administration.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the double-stranded RNAi agent or a salt thereof is administered to the human subject via subcutaneous administration.

13. The pharmaceutical composition according to claim 12, wherein the subcutaneous administration is self-administered.

14. The pharmaceutical composition according to claim 13, wherein the self-administration is performed via a pre-filled syringe or an auto-injector syringe.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the level of TTR mRNA expression or TTR protein expression in the sample derived from the human subject is evaluated.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the double-stranded RNAi agent or a salt thereof is administered to the human subject every three months, every four months, every five months, every six months, every nine months, or every twelve months.

17. The pharmaceutical composition according to any one of claims 1 to 15, wherein the double-stranded RNAi agent or a salt thereof is administered to the human subject once every three months in the aforementioned fixed dose.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the double-stranded RNAi agent or a salt thereof is administered chronically to the human subject.

19. The pharmaceutical composition according to any one of claims 1 to 18, further comprising an additional therapeutic agent.

20. The pharmaceutical composition according to claim 19, wherein the additional therapeutic agent is a TTR tetramer stabilizer and / or a nonsteroidal anti-inflammatory agent.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand.

22. The pharmaceutical composition according to claim 21, wherein the ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

23. The ligand is 【Chemistry 1】 The pharmaceutical composition according to claim 21.

24. The pharmaceutical composition according to claim 21, wherein the ligand is bound to the 3' end of the sense chain.

25. The aforementioned double-stranded RNAi agent is shown in the following schematic diagram. 【Chemistry 2】 (In the formula, X is either O or S) The pharmaceutical composition according to claim 24, conjugated to the ligand shown in [reference].

26. The sense strand of the double-stranded RNAi agent comprises the nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' of SEQ ID NO: 10, and the antisense strand of the double-stranded RNAi agent comprises the nucleotide sequence 5'-usCfsuugGfuuAfcaugAfaAfucccasusuc-3' of SEQ ID NO: 7, Here, a, c, g, and u are 2'-O-methyl(2'-OMe)A, C, G, or U; Af, Cf, Gf, and Uf are 2'-fluoroA, C, G, or U; s is a phosphorothioate bond; and The aforementioned double-stranded RNAi agent is shown in the following schematic diagram. 【Transformation 3】 (In the equation, X is O) A pharmaceutical composition according to any one of claims 1 to 25, conjugated to a ligand shown in [the specified figure].

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