KETOHEXOKINASE (KHK) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

To provide methods of treating subjects having a ketohexokinase (KHK)-associated disorder, e.g., liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), disorders of glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney disorder, tubular dysfunction, proinflammatory changes to the proximal tubules), metabolic syndrome, adipocyte dysfunction, visceral adipose deposition, obesity, hyperuricemia, gout, eating disorders, and excessive sugar craving.SOLUTION: The present invention provides a double stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on February 11, 2015, is titled 121301-01220_SL.txt and is 116,205 bytes in size.

[0002] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 938,567, filed February 11, 2014, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Epidemiological studies have shown that the Western diet is one of the main causes of the recent global obesity epidemic. Increased fructose intake, associated with the use of fortified soft drinks and processed foods, has been proposed as a major factor in this epidemic. High-fructose corn syrup began to be widely used in the food industry by 1967. Although glucose and fructose have the same caloric value per molecule, these two sugars are metabolized differently and use different GLUT transporters. Fructose is metabolized almost exclusively in the liver, and unlike the glucose metabolic pathway, the fructose metabolic pathway is not regulated by product feedback inhibition (Non-Patent Document 1). While hexokinase and phosphofructokinase (PFK) regulate the production of glyceraldehyde-3-P from glucose, fructokinase or ketohexokinase (KHK), which is involved in the phosphorylation of fructose to fructose-1-phosphate in the liver, is downregulated by increased fructose-1-phosphate concentrations. As a result, all fructose entering cells is rapidly phosphorylated (Non-Patent Document 2). The continuous use of ATP to phosphorylate fructose to fructose-1-phosphate leads to intracellular phosphate depletion, ATP depletion, activation of AMP deaminase, and the formation of uric acid (Non-Patent Document 1). Increased uric acid further promotes the upregulation of KHK (Non-Patent Document 3), causing endothelial cell and adipocyte dysfunction. Subsequently, fructose-1-phosphate is converted to glyceraldehyde by the action of aldolase B and phosphorylated to glyceraldehyde-3-phosphate. Glyceraldehyde-3-phosphate proceeds downstream in the glycolytic pathway to form pyruvate, which enters the citric acid cycle, from which, under nutrient-sufficient conditions, citrate is transported from mitochondria to the cytosol to provide acetyl-coenzyme A for lipogenesis (Figure 1).

[0004] Fructose phosphorylation by KHK and subsequent activation of lipogenesis leads to, for example, fatty liver, hypertriglyceridemia, dyslipidemia, and insulin resistance. It has also been shown that inflammatory changes in proximal tubule cells are induced by KHK activity (Non-Patent Document 2). Fructose phosphorylation by KHK is associated with diseases, disorders, and / or conditions such as liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, renal tubular dysfunction, proximal tubule inflammatory changes), metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar craving. Thus, there is a need in the art for compositions and methods for treating diseases, disorders, and / or conditions associated with KHK activity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Khaitan Z et al.,(2013)J.Nutr.Metab.2013,Article ID 682673,1-12 [Non-patent document 2] Cirillo P. et al.,(2009)J.Am.Soc.Nephrol.20:545-553 [Non-patent document 3] Lanaspa MAet al.,(2012)PLOS ONE 7(10):1-11 Summary of the Invention [Means for solving the problem]

[0006] The present invention provides compositions comprising RNAi agents, e.g., double-stranded RNAi agents, that target ketohexokinase (KHK). The present invention also provides methods of using the compositions of the present invention to inhibit KHK expression and / or to treat disorders that may benefit from inhibiting or reducing KHK gene expression, such as KHK-related diseases, such as liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubule), metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar craving.

[0007] Thus, in one aspect, the present invention provides a double-stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by no more than 3 nucleotides.

[0008] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), comprising a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differs from any one of the antisense sequences listed in any one of Tables 3, 4, and 5 by no more than 3 nucleotides.

[0009] In one embodiment, the sense strand and the antisense strand comprise a sequence selected from the group consisting of AD-63851, AD-63820, AD-63853, AD-63839, AD-63854, AD-63855, and AD-63886, and any one of the sequences disclosed in any one of Tables 3, 4, 8, 11, 12, 14, and 15.

[0010] In one embodiment, the double-stranded RNAi agent comprises at least one modified nucleotide.

[0011] In one embodiment, the at least one modified nucleotide is a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a terminal nucleotide attached to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide. The nucleotide is selected from the group consisting of nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0012] In another embodiment, the at least one modified nucleotide is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0013] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by no more than 3 nucleotides, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at its 3' end.

[0014] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0015] In another embodiment, the sense and antisense strands comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, and 7.

[0016] In certain embodiments, the modified nucleotide is a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a terminal nucleotide attached to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide. , 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0017] In another embodiment of the double-stranded RNAi agent, at least one strand comprises a 3' overhang of at least one nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides.

[0018] In yet another embodiment, the double-stranded RNAi agent comprises a ligand. In another embodiment, the double-stranded RNAi agent is administered using a pharmaceutical composition and a lipid formulation. In another embodiment, the lipid formulation comprises lipid nanoparticles (LNPs). In yet another embodiment, the lipid nanoparticles (LNPs) comprise MC3 lipids.

[0019] In another aspect, the invention provides a composition comprising a modified antisense polynucleotide agent, the agent being capable of inhibiting expression of ketohexokinase (KHK) in a cell and comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 3, 4, and 5, wherein the polynucleotide is about 14 to about 30 nucleotides in length.

[0020] In another aspect, the present invention provides an RNAi agent capable of inhibiting expression of ketohexokinase (KHK) in a cell, e.g., a double-stranded RNAi agent, comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; Each n may or may not be present p , n p ',n q , and n q ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; N b The modification on Y is different from the modification on N b the modification on Y' is different from the modification on Y'; and the sense strand is conjugated to at least one ligand The present invention provides a double-stranded RNAi agent represented by:

[0021] In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1.

[0022] In one embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0023] In one embodiment, the YYY motif is present at or near the cleavage site on the sense strand.

[0024] In another embodiment, the Y'Y'Y' motif is present at positions 11, 12, and 13 of the antisense strand from the 5' end.

[0025] In one embodiment, Y' is 2'-O-methyl.

[0026] In one embodiment, formula (III) is (IIIa): Sense:5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5'(IIIa) is expressed by

[0027] In another embodiment, formula (III) is formula (IIIb): Sense:5'n p -N a -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIIb) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) is expressed by

[0028] In yet another embodiment, formula (III) is formula (IIIc): Sense:5'n p -N a -XXX-N b -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5'(IIIc) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) is expressed by

[0029] In a further embodiment, formula (III) is formula (IIId): Sense:5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIId) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N a and N a ' independently represent an oligonucleotide sequence containing 2 to 10 modified nucleotides) is expressed by

[0030] In one embodiment, the double-stranded region is 15 to 30 nucleotide pairs in length. In another embodiment, the double-stranded region is 17 to 23 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 17 to 25 nucleotide pairs in length. In a further embodiment, the double-stranded region is 23 to 27 nucleotide pairs in length. In another embodiment, the double-stranded region is 19 to 21 nucleotide pairs in length. In another embodiment, the double-stranded region is 19 to 23 nucleotide pairs in length. In another embodiment, the double-stranded region is 21 to 23 nucleotide pairs in length. In yet another embodiment, each strand has 15 to 30 nucleotides.

[0031] In one embodiment, the modification on the nucleotide is selected from the group consisting of LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In another embodiment, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0032] In one embodiment, the Ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. In another embodiment, the Ligand is [ka] is.

[0033] In one embodiment, the ligand is attached to the 3' end of the sense strand.

[0034] In another embodiment, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S. In certain embodiments, X is O.

[0035] In one embodiment, the agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0036] In a further embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand. In another embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0037] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. In another embodiment, the strand is the antisense strand. In a further embodiment, the strand is the sense strand.

[0038] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand, hi another embodiment, the strand is the antisense strand.

[0039] In another embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In a further embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-end and two phosphorothioate internucleotide linkages at the 3'-end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5'-end or the 3'-end of the sense strand.

[0040] In one embodiment, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair. In another embodiment, the Y nucleotide comprises a 2'-fluoro modification. In a further embodiment, the Y' nucleotide comprises a 2'-O-methyl modification.

[0041] In one embodiment, p'>0. In another embodiment, p'=2. In a further embodiment, q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA. In yet another embodiment, q'=0, p=0, q=0, and the p' overhanging nucleotides are non-complementary to the target mRNA.

[0042] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0043] In another embodiment, at least one n pIn a further embodiment, all n' are linked to adjacent nucleotides via phosphorothioate bonds. p ' is attached to the adjacent nucleotide via a phosphorothioate bond.

[0044] In another embodiment, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3, 4, and 5.

[0045] In one aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of ketohexokinase (KHK). The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, wherein substantially all of the nucleotides of the sense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and substantially all of the nucleotides of the antisense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus, and wherein the sense strand is conjugated at the 3'-terminus to one or more GalNAc derivatives attached via a branched bivalent or trivalent linker.

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

[0047] In another aspect, the present invention provides an RNAi agent, for example, a double-stranded RNAi agent, capable of inhibiting the expression of KHK (ketohexokinase) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, and the antisense strand comprises a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; Each n may or may not be present p , n p ',n q , and n q ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b the modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand is conjugated to at least one ligand The present invention provides a double-stranded RNAi agent represented by:

[0048] In yet another aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of ketohexokinase (KHK) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b the modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand is conjugated to at least one ligand The present invention provides a double-stranded RNAi agent represented by:

[0049] In a further aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of ketohexokinase (KHK) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b the modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; The present invention provides a double-stranded RNAi agent represented by:

[0050] In another aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of ketohexokinase (KHK) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b the modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand comprises at least one phosphorothioate linkage; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; The present invention provides a double-stranded RNAi agent represented by:

[0051] In yet another aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of ketohexokinase (KHK) in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-n q '5'(IIIa) (In the formula: Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; the sense strand comprises at least one phosphorothioate linkage; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; The present invention provides a double-stranded RNAi agent represented by:

[0052] The present invention also provides cells, vectors, host cells and pharmaceutical compositions comprising the double-stranded RNAi agents of the present invention.

[0053] In one embodiment, the invention provides a double-stranded RNAi agent comprising an RNAi agent listed in any one of Tables 3, 4 5, 6, and 7.

[0054] In one embodiment, the cell comprises a double-stranded RNAi agent.

[0055] In another embodiment, the vector encodes at least one strand of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a region of complementarity to at least a portion of an mRNA encoding ketohexokinase, the double-stranded RNAi agent being 30 base pairs or less in length, and the double-stranded RNAi agent targets the mRNA for cleavage. In a further embodiment, the region of complementarity is at least 15 nucleotides in length. In another embodiment, the region of complementarity is 19-21 nucleotides in length. In another embodiment, a cell comprises the vector.

[0056] In certain embodiments, the composition comprising the double-stranded RNAi agent or modified antisense polynucleotide agent is administered using a pharmaceutical composition.

[0057] In a preferred embodiment, the double-stranded RNAi agent is administered in a solution. In one embodiment, the double-stranded RNAi agent is administered in an unbuffered solution. In another embodiment, the unbuffered solution is saline or water. In another embodiment, the double-stranded RNAi agent is administered with a buffer. In yet another embodiment, the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS).

[0058] In another aspect, the present invention provides a method for inhibiting ketohexokinase (KHK) expression in a cell, the method comprising contacting the cell with a double-stranded RNAi agent, a pharmaceutical composition, a composition comprising a modified antisense polynucleotide agent, or a vector comprising an RNAi agent, and maintaining the resulting cells for a time sufficient to allow degradation of mRNA transcripts of the KHK gene, thereby inhibiting expression of the KHK gene in the cell.

[0059] In one embodiment, the cell is in a subject. In a further embodiment, the subject is human. In a further embodiment, the subject is suffering from a disease associated with ketohexokinase.

[0060] In one embodiment, KHK expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0061] In another aspect, the invention provides a method of treating a subject suffering from a ketohexokinase (KHK)-associated disorder, the method comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, a composition comprising a modified antisense polynucleotide agent, or a pharmaceutical composition comprising a double-stranded RNAi agent, thereby treating the subject.

[0062] In another aspect, the invention provides a method of treating a subject suffering from a disorder associated with ketohexokinase (KHK), comprising the step of subcutaneously administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, and wherein substantially all of the nucleotides in the antisense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus, substantially all of the nucleotides in the sense strand comprise modifications selected from the group consisting of 2'-O-methyl and 2'-fluoro modifications, the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus, and the sense strand is conjugated at the 3'-terminus to one or more GalNAc derivatives linked via a branched bivalent or trivalent linker.

[0063] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand comprise a modification.

[0064] In one embodiment, the subject is a human.

[0065] In one embodiment, the disease associated with ketohexokinase is selected from the group consisting of liver disease, dyslipidemia, impaired glycemic control, cardiovascular disease, kidney disease, metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar craving. In a specific embodiment, the liver disease is fatty liver and / or steatohepatitis. In another embodiment, the dyslipidemia is selected from the group consisting of hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, and postprandial hypertriglyceridemia. In yet another embodiment, the impaired glycemic control is insulin resistance and / or diabetes. In a further embodiment, the cardiovascular disease is hypertension and / or endothelial cell dysfunction. In yet another embodiment, the kidney disease is selected from the group consisting of acute kidney injury, renal tubular dysfunction, and inflammatory changes in the proximal tubule.

[0066] In one embodiment, the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 1 mg / kg to about 10 mg / kg. In a preferred embodiment, the double-stranded RNAi agent is administered at a dose of about 0.1 mg / kg, about 1.0 mg / kg, or about 3.0 mg / kg. In a specific embodiment, the double-stranded RNAi agent is administered at a dose of about 1 mg / kg to about 10 mg / kg.

[0067] In one embodiment, the double-stranded RNAi agent is administered subcutaneously or intravenously.

[0068] In one embodiment, the RNAi agent is administered in two or more doses.

[0069] In yet another embodiment, the method further comprises administering to the subject an additional therapeutic agent, hi some embodiments, the additional therapeutic agent is selected from the group consisting of an HMG-CoA reductase inhibitor, an antidiabetic agent, an antihypertensive agent, and resveratrol. [Brief explanation of the drawings]

[0070] [Figure 1] 1 shows the metabolism of fructose by ketohexokinase and the metabolism of glucose and fructose by hexokinase. [Figure 2] The exon arrangement in the human KHK gene for the ketohexokinase A transcript (NM_000221.2), the ketohexokinase C transcript (NM_006488.2), and the transcript variant X5 (XM_005264298.1) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0071] The invention provides compositions that include RNAi agents, eg, double-stranded iRNA agents, that target KHK. The present invention also provides methods of using the compositions of the present invention to inhibit KHK expression and treat KHK-related diseases, disorders, and / or conditions, such as liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubule), metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings (Khaitan Z. et al., (2013) J. Nutr. Metab., Article ID 682673, 1-12; Diggle CPet al.,(2009)J.Hisotchem.Cytochem.,57(8):763-774;Cirillo P.et al.,(2009)J.Am.Soc.Nephrol.,20:545-553;Lanaspa MAet al.,(2012)PLOS ONE 7(10):1-11).

[0072] The KHK (ketohexokinase) gene, located on chromosome 2p23, encodes ketohexokinase, also known as fructokinase. KHK is a phosphotransferase enzyme that uses alcohol as a phosphate acceptor. KHK belongs to the ribokinase family of carbohydrate kinases (Trinh et al., ACTA Cryst., D65:201-211). Two isoforms of ketohexokinase, KHK-A and KHK-C, have been identified and are derived from alternative splicing of the full-length mRNA. These isoforms differ in the inclusion of either exon 3a or 3c and differ by 32 amino acids at positions 72-115 (see, for example, Figure 2). KHK-C mRNA is expressed at high levels primarily in the liver, kidney, and small intestine. KHK-C has a much lower K for fructose binding than KHK-A. m As a result, it is highly effective in phosphorylating dietary fructose. The sequence of human KHK-C mRNA transcript can be found, for example, in GenBank accession number GI:153218447 (NM_006488.2; SEQ ID NO:1). The sequence of human KHK-A mRNA transcript can be found, for example, in GenBank accession number GI:153218446 (NM_000221.2; SEQ ID NO:3). The sequence of full-length human KHK mRNA is provided in GenBank accession number GI:530367552 (XM_005264298.1; SEQ ID NO:5) and was used (Figure 2).

[0073] The present invention provides iRNA agents, compositions, and methods for regulating KHK gene expression. In certain embodiments, KHK expression is reduced or inhibited using a KHK-specific iRNA agent, thereby reducing the phosphorylation of fructose to fructose-1-phosphate, thereby preventing increased uric acid levels and increased lipogenesis. Thus, inhibiting KHK gene expression or activity using the iRNA compositions of the invention is useful as a therapeutic method for reducing the lipogenic effects of dietary fructose and preventing the associated accumulation of uric acid in a subject. Such inhibition is useful for treating diseases, disorders, and / or conditions such as liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, renal tubular dysfunction, inflammatory changes in the proximal tubule), metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings.

[0074] I. Definition So that the present invention may be more readily understood, several terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a variable is recited, all values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0075] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0076] The term "including" is used herein to mean, and is used synonymously with, the phrase "including but not limited to."

[0077] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0078] As used herein, "KHK" refers to the ketohexokinase gene or protein. KHK is also known as fructokinase. The term "KHK" includes human KHK, the amino acid and complete coding sequence of which can be found, for example, in GenBank accession number BC006233. The sequence of human KHK-C mRNA transcript can be found, for example, in GenBank accession number GI:153218447 (NM_006488.2; SEQ ID NO:1). The reverse complement of SEQ ID NO:1 is provided as SEQ ID NO:2. The sequence of human KHK-A mRNA transcript can be found, for example, in GenBank accession number GI:153218446 (NM_000221.2; SEQ ID NO:3). The reverse complement of SEQ ID NO:3 is provided as SEQ ID NO:4. The sequence of the human full-length KHK mRNA transcript is provided in GenBank Accession No. GI:530367552 (XM_005264298.1; SEQ ID NO:5). The reverse complement of SEQ ID NO:5 is provided as SEQ ID NO:6. The sequence of mouse (Mus musculus) KHK mRNA can be found, for example, in GenBank Accession No. GI:118130797 (NM_008439.3; SEQ ID NO:7), with the reverse complement sequence provided as SEQ ID NO:8. The sequence of rat (Rattus rattovorus) KHK mRNA can be found, for example, in GenBank Accession No. GI:126432547 (NM_031855.3; SEQ ID NO:9), with the reverse complement sequence provided as SEQ ID NO:10. The sequence of cynomolgus monkey (Macaca fascicularis) KHK mRNA, variant X1 can be found, for example, in GenBank Accession No. GI:544482340 (XM_005576321.1; SEQ ID NO: 11) or GI:, with the reverse complement sequence provided in SEQ ID NO: 12. The sequence of cynomolgus monkey (Macaca fascicularis) KHK mRNA, variant X3 can be found, for example, in GenBank Accession No. GI:544482340 (XM_005576321.1; SEQ ID NO: 325) or GI:, with the reverse complement sequence provided in SEQ ID NO: 326.Further examples of KHK mRNA sequences are readily available using public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0079] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of a KHK gene, including mRNA that is the product of RNA processing of a primary transcript.

[0080] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of a KHK gene, including mRNAs that are the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed upon transcription of a KHK gene.

[0081] The target sequence can be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence can be about 15 to 30 nucleotides, 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, 1 It can be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also considered part of the invention.

[0082] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by a sequence given using standard nucleotide nomenclature.

[0083] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or surrogate replacement moieties, as described in more detail below (see, for example, Table 2). Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil may be substituted by other moieties without significantly changing the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine may be substituted, for example, by a nucleotide containing inosine in the nucleotide sequence of a dsRNA characterized in the present invention. In another example, adenine and cytosine anywhere in an oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in the present invention.

[0084] The terms "iRNA," "RNAi agent," "iRNA agent," "RNA interfering agent," or "small interfering RNA" or "siRNA," as used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as those terms are defined herein. iRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (iRNA). iRNA regulates (e.g., inhibits) expression of KHK in cells, e.g., cells in a subject, such as a mammalian subject.

[0085] In one embodiment, the iRNA agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, e.g., a KHK target mRNA sequence, and directs cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 base pair short interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Then, siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to the appropriate target mRNA, one or more endonucleases in RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Here, in one aspect, the present invention relates to a single-stranded RNA (siRNA) that is produced in cells and promotes the formation of a RISC complex that leads to the silencing of the target gene, i.e., the KHK gene. Therefore, the term "siRNA" is also used herein to refer to the above-mentioned iRNA.

[0086] In another embodiment, an iRNA agent can be a single-stranded siRNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded iRNA agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs is described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs chemically modified as described herein or by the methods described in Lima et al., (2012) Cell 150;:883-894.

[0087] In another embodiment, the "iRNA" for use in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which are shown to have "sense" and "antisense" orientations relative to the target RNA, i.e., the KHK gene. In certain embodiments of the present invention, the double-stranded RNA (dsRNA) causes degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference, or RNAi, or iRNA.

[0088] Generally, the majority of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, although, as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, an "iRNA agent" can include ribonucleotides with chemical modifications; an iRNA agent can include substantial modifications at multiple nucleotides.

[0089] As used herein, the term "modified nucleotide" refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide bond, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses, for example, the substitution, addition, or removal of a functional group or atom to an internucleoside bond, a sugar moiety, or a nucleobase. Modifications suitable for use in the agents of the present invention include any type of modification disclosed herein or known in the art. Any such modifications when used in siRNA-type molecules are encompassed by "RNAi agent" for the purposes of this specification and claims.

[0090] The double-stranded region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can range from about 9 to 36 base pairs in length, e.g., about 15 to 30 base pairs in length, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27 , 19-26, 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 The nucleic acid sequence may be about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the invention.

[0091] The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore connected by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand that form the double-stranded structure, the connected RNA strands are called "hairpin loops." A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 23, or more unpaired nucleotides.

[0092] When the two substantially complementary strands of a dsRNA are constituted by separate RNA molecules, the molecules can be, but do not have to be, covalently linked.

[0093] When two strands are covalently linked by means other than a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand that form the double-stranded structure, the linked structure is called a "linker." The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus the overhangs present in the double strand. In addition to the double-stranded structure, the iRNA agent can include one or more nucleotide overhangs.

[0094] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be in the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be present at the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0095] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end and / or 5'-end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end and / or 5'-end. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0096] "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at the relevant end of a double-stranded iRNA agent, i.e., there are no nucleotide overhangs. A "blunt-ended" iRNA agent is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. iRNA agents of the invention include iRNA agents with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or RNAi agents with nucleotide overhangs at both ends.

[0097] The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., a KHK mRNA. As used herein, the term "region of complementarity," as defined herein, refers to a region of the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a KHK nucleotide sequence. When the region of complementarity is not perfectly complementary to the target sequence, mismatches can occur in internal or terminal regions of the molecule. Generally, most tolerated mismatches occur in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the iRNA.

[0098] The term "sense strand," or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0099] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site in the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0100] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a double-stranded structure under defined conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may occur inside an organism, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0101] A complementary sequence in an iRNA, e.g., a dsRNA, described herein includes base pairing across the entire length of one or both nucleotide sequences of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than five, four, three, or two mismatched base pairs upon hybridization to a duplex of up to 30 base pairs while retaining the ability to hybridize under conditions optimal for their ultimate application, e.g., inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, for purposes described herein, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length may be referred to as "fully complementary" if the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide.

[0102] As used herein, "complementary" sequences can also include, or be formed entirely of, non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairs.

[0103] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to matching bases between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood from the context of their use.

[0104] As used herein, a polynucleotide "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding KHK), including the 5' UTR, open reading frame (ORF), or 3' UTR. For example, a polynucleotide is complementary to at least a portion of a KHK mRNA if its sequence is substantially complementary to a contiguous portion of an mRNA encoding KHK.

[0105] Generally, the majority of the nucleotides in each strand are ribonucleotides; however, as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. Furthermore, an "iRNA" may contain ribonucleotides having chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any such modifications when used in an iRNA molecule are encompassed by "iRNA" for purposes of this specification and claims.

[0106] In one embodiment of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base pairing to the mRNA and physically interfering with the translation machinery (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense oligonucleotide molecule can be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can contain a sequence that is at least about 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from any one of the antisense sequences described herein.

[0107] As used herein, the term "inhibiting" is used interchangeably with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes any level of inhibition.

[0108] As used herein, the phrase "inhibiting the expression of KHK" includes the inhibition of the expression of any KHK gene (e.g., mouse KHK gene, rat KHK gene, monkey KHK gene, or human KHK gene, etc.) as well as mutants or variants of the KHK gene that encode KHK proteins.

[0109] "Inhibiting expression of the KHK gene" includes any level of inhibition of the KHK gene, for example, at least partial suppression of expression of the KHK gene, such as 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%, or at least about 99% inhibition.

[0110] Expression of the KHK gene can be evaluated based on the level of any variable associated with KHK gene expression, such as KHK mRNA level or KHK protein level. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level measured from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0111] In one embodiment, at least partial suppression of KHK gene expression is assessed by a reduction in the amount of KHK mRNA that can be isolated from or detected in a first cell or group of cells in which the KHK gene is transcribed and that have been treated to inhibit KHK gene expression, compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been so treated. The degree of inhibition can be expressed by the following formula:

number

[0112] As used herein, the phrase "contacting a cell with an iRNA agent (such as a dsRNA)" includes contacting a cell by any possible means. Contacting a cell with an iRNA agent includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo. Contacting can be direct or indirect. Thus, for example, an RNAi agent can be physically contacted with a cell by performing a method individually, or the iRNA agent can be placed in a situation that allows or causes it to later contact a cell.

[0113] Contacting cells in vitro can be performed, for example, by incubating the cells with an iRNA agent. Contacting cells in vivo can be performed, for example, by injecting the iRNA agent into or near the tissue where the cells are located, or by injecting the iRNA agent into another area, such as the bloodstream or subcutaneous space, so that the iRNA agent will subsequently reach the tissue where the contacted cells are located. For example, the iRNA agent can contain and / or be bound to a ligand, e.g., GalNAc3, that directs the iRNA agent to a site of interest, e.g., the liver. A combination of in vitro and in vivo contacting methods is also possible. For example, cells can also be contacted with an iRNA agent in vitro and then transplanted into a subject.

[0114] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering the iRNA into a cell" by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the iRNA can occur by unassisted diffusion or active cellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved by β-glucan delivery systems, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677 and U.S. Patent Application Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Additional techniques are described herein below and / or are known in the art.

[0115] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically effective molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid into which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0116] As used herein, a "subject" is an animal such as a mammal, including a primate (such as a human or a non-human primate, e.g., a monkey or chimpanzee), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, and / or condition that may benefit from reduced expression of KHK as described herein; a human at risk for a disease, disorder, and / or condition that may benefit from reduced expression of ketohexokinase; or a human being treated for a disease, disorder, or condition that may benefit from reduced expression of ketohexokinase.

[0117] As used herein, the terms "treat" or "treatment" include, but are not limited to, one or more conditions associated with undesired ketohexokinase activation (e.g., liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, renal tubular dysfunction, inflammatory changes in the proximal tubule), metabolic syndrome (e.g., urinary insufficiency, urinary tract infection, urinary tract infection), and the like). "Treatment" refers to beneficial or desired results, including alleviation or amelioration of ketohexokinase syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings; stabilization (i.e., not worsening) of one or more diseases, disorders, and / or conditions associated with ketohexokinase activity; and improvement or amelioration of undesirable ketohexokinase activity, whether detectable or undetectable (e.g., fructose phosphorylation, which leads to activated lipogenesis and increased uric acid production). "Treatment" can also mean prolonging survival as compared to expected survival without treatment.

[0118] As a result, alleviation or improvement of any of the symptoms resulting from fructose metabolism via the KHK pathway includes alleviation or improvement of any one or more of the following symptoms, including, but not limited to, fatty liver, steatohepatitis, high blood pressure, hypertension, high cholesterol, high LDL cholesterol, low HDL cholesterol, hyperlipidemia, hypertriglyceridemia, kidney disease, metabolic syndrome, excessive sugar cravings, eating disorders, postprandial hypertriglyceridemia, hepatosteatosis, gout, diabetes, acute kidney injury, renal tubular dysfunction, insulin resistance, and obesity. In one embodiment, alleviation or improvement of a KHK-related disease, disorder, or condition refers to alleviation of fatty liver, excess body fat, obesity, high cholesterol, hypertension, or high blood pressure.

[0119] The term "reducing," in reference to the level of ketohexokinase activity in a subject, or as a disease marker or symptom, refers to a statistically significant decrease in such level, which may be, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, preferably to a level considered to be within the normal range for individuals without such disorder.

[0120] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that may benefit from reduced expression of a KHK gene, refers to a reduced likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, renal tubular dysfunction, inflammatory changes in the proximal tubule), metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and symptoms of the phosphorylation of fructose to fructose-1-phosphate, such as, for example, activated lipogenesis and increased production of uric acid, which lead to excessive sugar cravings. The likelihood of developing any or all of these diseases, disorders, and / or conditions is reduced, for example, if an individual who has one or more risk factors for any or all of these diseases, disorders, and / or conditions either does not develop the disease, disorder, and / or condition, or develops a milder form of the disease, disorder, and / or condition compared to a population with the same risk factors who does not receive the treatment described herein. Failure to develop the disease, disorder, and / or condition, or a reduction in the occurrence of symptoms associated with such disease, disorder, and / or condition (e.g., by at least about 10% on a clinically recognized measure of the disease or disorder), or a delayed onset of symptoms (e.g., by only days, weeks, months, or years) is considered effective prevention.

[0121] As used herein, the term "ketohexokinase-associated disease" refers to a disease, disorder, or condition caused by or associated with a ketohexokinase gene or protein, such as a disease, disorder, or condition caused by or associated with the phosphorylation of fructose to fructose-1-phosphate. Such diseases are typically associated with activated lipogenesis and increased uric acid production. Non-limiting examples of diseases associated with ketohexokinase include liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), abnormal glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubule), metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings.

[0122] As used herein, a "therapeutically effective amount" is intended to include an amount of an iRNA agent that, when administered to a subject suffering from a disease or disorder associated with KHK, is sufficient to treat the disease or disorder (e.g., by reducing or ameliorating one or more symptoms of the disease or disorder). A "therapeutically effective amount" can vary depending on the RNAi agent, the manner in which the agent is administered, the disease and its severity, and the subject being treated's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics.

[0123] As used herein, a "prophylactically effective amount" is intended to include an amount of an iRNA agent sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject suffering from a KHK-associated disease and / or a subject at risk for developing a KHK-associated disease, e.g., liver disease, dyslipidemia, impaired glycemic control, cardiovascular disease, kidney disease, metabolic syndrome, and / or obesity, but who has not yet (or is not currently) experiencing or exhibiting symptoms of the disease. Amelioration of the disease includes slowing the course of the disease or reducing the severity of later-onset disease. A "prophylactically effective amount" can vary depending on the iRNA agent, the manner in which the agent is administered, the risk of acquiring the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics being treated.

[0124] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces a desired local or systemic effect at a reasonable benefit-to-risk ratio applicable to any treatment. The iRNA agent used in the methods of the invention can be administered in an amount sufficient to obtain a reasonable benefit-to-risk ratio applicable to such treatment. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human and animal subjects without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-to-risk ratio.

[0125] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound to a subject from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) celluloses and their derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free distilled water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (22) other non-toxic, compatible materials used in pharmaceutical formulations.

[0126] As used herein, the term "sample" includes similar body fluids, cells, or tissues isolated from a subject, as well as collections of body fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, urine, lymph, cerebrospinal fluid, ocular fluid, saliva, etc. Tissue samples can include samples derived from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or body fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells in the liver, such as hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma obtained from a subject. In further embodiments, a "sample derived from a subject" refers to liver tissue (or a subcomponent thereof) obtained from a subject.

[0127] II. iRNAs of the Invention The present invention also provides iRNAs that inhibit expression of the KHK gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the KHK gene in cells, such as cells in a mammal, e.g., a human, suffering from a disease or disorder associated with KHK, including, but not limited to, liver disease (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular disease (e.g., hypertension, endothelial cell dysfunction), kidney disease (e.g., acute kidney injury, renal tubular dysfunction, inflammatory changes in the proximal tubule), metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar craving. The dsRNA comprises an antisense strand having a region complementary to at least a portion of the mRNA formed during expression of the KHK gene. The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing a KHK gene, the iRNA inhibits expression of the KHK gene (e.g., a human, primate, non-primate, or avian KHK gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as, for example, by immunofluorescence analysis using Western blot or flow cytometry techniques.

[0128] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the target sequence, generally completely complementary. The target sequence can be derived from the sequence of the mRNA formed during the expression of the KHK gene. The other strand (sense strand) comprises a region complementary to the antisense strand, so that the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere herein and known in the art, the complementary sequence of the dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, rather than being on a separate oligonucleotide.

[0129] Generally, the double-stranded structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 1 21-23, 21-22, or 21-22 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present invention.

[0130] Similarly, the region of complementarity to the target sequence may be 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-40, 19-50, 19-60, 19-70, 19-80, 19-90, 19-100, 19-110, 19-120, 19-130, 19-140, 19-150, 19-210, 19-220, 19-230, 19-240, 19-250, 19-260, 19-270, 19-280, 19-290, 19-300, 19-310, 19-320, 19-330, 19-340, 19-350, 19-360, 19-370, 19-380, 19-410, 19-420, 19-430, 19-440, 19-450, 19-460, 19-470, 19-480, 19-510, 21-23, 21-22, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present invention.

[0131] In certain embodiments, the dsRNA is about 15 to about 20 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21 to 23 nucleotides in length can serve as substrates for Dicer. As those skilled in the art will also recognize, the region of RNA targeted for cleavage is most often a portion of a larger RNA molecule (often an mRNA molecule). Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for iRNA-directed cleavage (i.e., cleavage via the RISC pathway).

[0132] The double-stranded region may be a primary functional portion of the dsRNA, e.g., about 9-36 base pairs, e.g., about 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, 9-3 3, 10-33, 11-33, 12-33, 13-33, 14-33, 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, 15-2 4, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 1 Those skilled in the art will also recognize that a double-stranded region of 9-22, 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 is also a dsRNA. In one embodiment, an RNA molecule or a complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, to the extent that it is processed into a functional double-strand of, for example, 15-30 base pairs that targets a desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting expression of KHK is not generated in the target cell by cleavage of a larger dsRNA.

[0133] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0134] dsRNA can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, Inc.), as further described below.

[0135] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA iRNA compounds can be prepared using solution phase or solid phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing unnatural or modified nucleotides can be easily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis, or both.

[0136] 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 group of sequences shown in any one of Tables 3, 4, 5, 6, and 7, and the corresponding antisense strand of the sense strand is selected from the group of sequences shown in any one of Tables 3, 4, 5, 6, and 7. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the mRNA produced during the expression of the KHK gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, where one oligonucleotide is represented as the sense strand in any one of Tables 3, 4, 5, 6, and 7, and the second oligonucleotide is represented as the corresponding antisense strand of the sense strand in any one of Tables 3, 4, 5, 6, and 7. In one embodiment, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.

[0137] Those skilled in the art are well aware that dsRNAs with a double-stranded structure of about 20-23 base pairs, for example, 21 base pairs, have been found to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others skilled in the art have found that shorter or longer RNA double-stranded structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above embodiment, depending on the nature of the oligonucleotide sequence shown in any one of Tables 3, 4, 5, 6, and 7, the dsRNAs described herein may comprise at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences shown in any one of Tables 3, 4, 5, 6, and 7, minus only a few nucleotides at one or both ends, may be similarly effective compared to the above-mentioned dsRNAs. Thus, dsRNAs having at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotide sequences from one of the sequences shown in any one of Tables 3, 4, 5, 6, and 7, whose ability to inhibit expression of the KHK gene differs from that of a dsRNA containing the complete sequence by no more than about 5, 10, 15, 20, 25, or 30% inhibition, are considered to be within the scope of the present invention.

[0138] Additionally, RNAs set forth in any one of Tables 3, 4, 5, 6, and 7 identify sites in the KHK transcript that are susceptible to RISC-mediated cleavage. Accordingly, the present invention also features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a specific site in an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that specific site. Such iRNAs will generally comprise at least about 15 contiguous nucleotides from one of the sequences set forth in any one of Tables 3, 4, 5, 6, and 7, linked to additional nucleotide sequences taken from regions adjacent to the selected sequence in the KHK gene.

[0139] Target sequences are generally approximately 15-30 nucleotides in length, although the suitability of specific sequences within this range for directing cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed literally or figuratively (including, for example, in silico) over the target RNA sequence to identify sequences within a size range that could serve as target sequences. The next potential target sequence can be identified by gradually shifting the sequence "window" one nucleotide upstream or downstream of the initial target sequence position until a complete set of possible sequences has been identified for any given target size selected. This process, along with systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, for example, while the sequences identified in any one of Tables 3, 4, 5, 6, and 7 represent effective target sequences, it is believed that further optimization of inhibitory efficiency may be achieved by gradually "moving the window" one nucleotide upstream or downstream of the given sequence to identify sequences with equivalent or better inhibitory properties.

[0140] Furthermore, for any sequence identified in any one of Tables 3, 4, 5, 6, and 7, further optimization could be achieved by systematically adding or removing nucleotides to generate longer or shorter sequences, and testing the resulting sequences by shifting the window of longer or shorter size from that point up or down the target RNA. Furthermore, coupling this approach to generating novel candidate targets with testing the efficacy of iRNAs based on those target sequences in inhibition assays known in the art and / or described herein could further improve the efficiency of inhibition. Furthermore, such optimized sequences could be adjusted, for example, by introducing modified nucleotides described herein or known in the art, adding or altering overhangs, or other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, improving transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

[0141] 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. If the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the region of mismatch is not located in the center of the region of complementarity. If the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 23-nucleotide iRNA agent, the strand complementary to a region of the KHK gene generally does not contain any mismatches within the central 13 nucleotides. Using methods described herein or known in the art, it can be determined whether an iRNA containing mismatches with the target sequence is effective in inhibiting KHK gene expression. Considering the effectiveness of iRNAs with mismatches in inhibiting KHK gene expression is important, especially when a particular region of complementarity in the KHK gene is known to have polymorphic sequence variation within the population.

[0142] III. Modified iRNAs of the Invention In one embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is unmodified, e.g., does not contain chemical modifications and / or conjugates known in the art and described herein. In another embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is chemically modified to improve stability or other beneficial properties. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA of the invention are modified. An iRNA of the invention in which "substantially all of the nucleotides are modified" is mostly, but not completely, modified and may contain no more than five, no more than four, no more than three, no more than two, or no more than one unmodified nucleotide.

[0143] Nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotide, inverted linkage, etc.); base modifications, such as substitution with a stable base, an unstable base, or a base that base-pairs with a wide range of partners, base removal (abasic nucleotide), or conjugated base; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and / or backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. The RNA with modified backbone particularly includes those that do not have a phosphorus atom in the backbone.For the purpose of this specification and as sometimes referred to in the art, the modified RNA that does not have a phosphorus atom in the internucleoside backbone can also be considered as oligonucleoside.In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0144] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates; thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters; and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0145] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,32 Specification No. 1,131; Specification No. 5,399,676; Specification No. 5,405,939; Specification No. 5,453,496; Specification No. 5,455,233; Specification No. 5,466,677; Specification No. 5,476,925 Specification No. 5,519,126; Specification No. 5,536,821; Specification No. 5,541,316; Specification No. 5,550,111; Specification No. 5,563,253; Specification No. 5,571,799; Specification No. 5,587 ,361 Specification; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534, Nos. 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0146] Modified RNA backbones that do not contain internal phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0147] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; Nos. 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0148] In other embodiments, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide units, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic, that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0149] Certain embodiments featured in the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly those described in U.S. Pat. No. 5,489,677, such as --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (also known as the methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--) and those described in U.S. Pat. No. 5,602,240, such as the amide backbones described in U.S. Pat. In certain embodiments, RNAs featured herein have the morpholino backbone structures described in U.S. Pat. No. 5,034,506, such as the morpholino backbone structures described in U.S. Pat.

[0150] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein, can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises at the 2' position a C1 to C 10The modification may include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacodynamic properties of iRNA, or group that improves the pharmacokinetic properties of iRNA, and other substituents with similar properties. In certain embodiments, the modification includes 2'-methoxyethoxy (2'-O--CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the Examples herein below.

[0151] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,81 Nos. 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, several of which are co-owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0152] iRNAs can also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), and 5-uracil (pseudouracil). Other synthetic and natural nucleobases include uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Concise Encyclopedia of Polymer Science and Engineering, pp. 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, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0153] Representative United States patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,1 Nos. 21, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0154] The RNA of an iRNA can also be modified to contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in certain embodiments, the agent of the present invention can contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety, where the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural configuration. The addition of a locked nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides containing a 4'-2' bridge.Examples of such 4'-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 "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,282). 83); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl), or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of these are incorporated herein by reference.

[0155] Further representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; Nos. 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0156] For example, any of the bicyclic nucleosides described above can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0157] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as an "S-cEt."

[0158] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation, increasing hybridization affinity for mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, reducing puckering of the ribose ring.

[0159] Representative publications that teach the preparation of some of the above-described CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383; and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0160] One or more of the nucleotides of the iRNA of the invention can also comprise a hydroxymethyl-substituted nucleotide, which is an acyclic 2'-3'-seco-nucleotide also known as an "unlocked nucleic acid" ("UNA") modification.

[0161] Representative U.S. patent publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. No. 8,314,227; and U.S. Patent Application Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference. Potentially stable modifications to the termini of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO 2011 / 005861.

[0162] A. Modified iRNAs Containing Motifs of the Invention In certain embodiments of the invention, double-stranded iRNA agents of the invention include agents having chemical modifications disclosed, for example, in U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, or PCT / U.S. Patent Application Publication No. 2010 / 065691, filed November 16, 2012, the entire contents of each of which are incorporated herein by reference.

[0163] As shown herein and in U.S. Provisional Patent Application No. 61 / 561,710 or PCT Application No. PCT / US2010 / 065691, better results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense and / or antisense strands of an iRNA agent, particularly at or near the cleavage site. In certain embodiments, the sense and antisense strands of an iRNA agent can be completely modified. Introduction of these motifs, if present, disrupts the modification pattern of the sense and / or antisense strands. The iRNA agent can optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand. The resulting iRNA agent exhibits better gene silencing activity.

[0164] More specifically, it has been surprisingly discovered that when the sense and antisense strands of a double-stranded iRNA agent are fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the iRNA agent, the gene silencing activity of the iRNA agent is significantly improved.

[0165] Thus, the present invention provides double-stranded iRNA agents capable of inhibiting expression of a target gene (i.e., a KHK gene) in vivo. The iRNA agent includes a sense strand and an antisense strand. Each strand of the iRNA agent can range from 12 to 30 nucleotides in length. For example, each strand can be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0166] The sense and antisense strands typically form double-stranded RNA ("dsRNA"), also referred to herein as an "iRNA agent" or "iRNA agent." The double-stranded region of an iRNA agent can be 12 to 30 nucleotide pairs in length. For example, the double-stranded region can be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0167] In one embodiment, an iRNA agent can include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs can form mismatches with the target mRNA, or the overhangs can be complementary to the targeted gene sequence, or they can be another sequence. The first and second strands can also be joined by additional bases or other non-basic linkers, e.g., to form a hairpin.

[0168] In one embodiment, each nucleotide in the overhang region of an iRNA agent can independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modification, such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be another sequence.

[0169] The 5'- or 3'-overhang on the sense strand, antisense strand, or both strands of an iRNA agent can be phosphorylated. In certain embodiments, the overhang region comprises two nucleotides with a phosphorothioate between them, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0170] An iRNA agent may contain only one overhang, which may enhance the interference activity of the iRNA without affecting its overall stability. For example, the single-stranded overhang may be located at the 3'-end of the sense strand or the 3'-end of the antisense strand. An iRNA 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 an iRNA has a nucleotide overhang at the 3'-end, and the 5'-end is blunt. Without wishing to be bound by theory, the asymmetric blunt ends at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favor the introduction of the guide strand into the RISC process.

[0171] In one embodiment, the iRNA agent is a 19-nucleotide double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 7, 8, and 9, from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0172] In another embodiment, the iRNA agent is a 20-nucleotide long, blunt-ended duplex, and the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 8, 9, and 10, from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0173] In yet another embodiment, the iRNA agent is a blunt-ended duplex 21 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 9, 10, and 11, from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0174] In one embodiment, the iRNA agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the iRNA agent is blunt while the other end comprises a two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of the three nucleotides being overhanging nucleotides and the third nucleotide being a pairing nucleotide adjacent to the overhanging nucleotide. In one embodiment, the iRNA agent further has two phosphorothioate internucleotide linkages between the terminal three nucleotides on 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 iRNA 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, e.g., in alternating motifs. Optionally, the iRNA agent further includes a ligand (preferably GalNAc3).

[0175] In one embodiment, the iRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1 to 23 of the sense strand to form a duplex; at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and the 5' end of the antisense strand is 10 to 30 consecutive nucleotides that are not paired with the sense strand. The antisense strand comprises at least one ribonucleotide at the 5'-end of the sense strand, thereby forming a 10-30 nucleotide single-stranded 5' overhang; at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so as to reduce expression of the target gene when the double-stranded nucleic acid is introduced into a mammalian cell; the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides, where at least one of the motifs is located at or near the cleavage site; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

[0176] In one embodiment, the iRNA agent includes a sense strand and an antisense strand, the iRNA agent including a first strand having a length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand including at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end; the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1-4 nucleotides longer than the first strand at its 3' end, the double-stranded region is at least 25 nucleotides long, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the second strand such that the iRNA agent reduces expression of the target gene when introduced into a mammalian cell, and dicer cleavage of the iRNA agent preferentially results in siRNA including the 3' end of the second strand, thereby reducing expression of the target gene in a mammal. Optionally, the iRNA agent further comprises a ligand.

[0177] In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs occurring at the cleavage site of the sense strand.

[0178] In one embodiment, the antisense strand of the iRNA agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs being at or near the cleavage site on the antisense strand.

[0179] In iRNA agents having a double-stranded region 17-23 nucleotides in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the iRNA from the 5' end.

[0180] The sense strand of iRNA agent may contain at least one motif of three identical modifications in three consecutive nucleotides at the site of strand breakage; the antisense strand may have at least one motif of three identical modifications in three consecutive nucleotides at or near the site of strand breakage.When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be aligned so that one motif of three nucleotides in the sense strand and one motif of three nucleotides in the antisense strand overlap by at least one nucleotide, that is, 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 overlap, or all three nucleotides may overlap.

[0181] In one embodiment, the sense strand of an iRNA agent can contain two or more motifs of three identical modifications in three consecutive nucleotides. The first motif can be at or near the cleavage site of the strand, and the other motifs can be wing modifications. The term "wing modification" herein refers to a motif that is present in another portion of the strand, away from a motif at or near the cleavage site of the same strand. The wing modifications can be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemical structures of the motifs are different from each other; when the motifs are separated by one or more nucleotides, the chemical structures can be the same or different. Two or more wing modifications can be present. For example, when two wing modifications are present, each wing modification can be at one end or on either side of the lead motif relative to the first motif at or near the cleavage site.

[0182] Like the sense strand, the antisense strand of an iRNA agent may contain two or more motifs of three identical modifications at three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage. The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present in the sense strand.

[0183] In one embodiment, wing modifications on the sense or antisense strand of an iRNA agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0184] In another embodiment, wing modifications on the sense or antisense strand of an iRNA agent typically do not include the first one or two paired nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0185] When the sense and antisense strands of an iRNA agent each include at least one wing modification, the wing modifications can be located at the same end of the double-stranded region and can have an overlap of 1, 2, or 3 nucleotides.

[0186] When the sense and antisense strands of an iRNA agent each include at least two wing modifications, the sense and antisense strands can be arranged so that two modifications from one strand are each located at one end of the double-stranded region, with an overlap of one, two, or three nucleotides; two modifications from one strand are each located at the other end of the double-stranded region, with an overlap of one, two, or three nucleotides; or two modifications from one strand are located on either side of the lead motif, with an overlap of one, two, or three nucleotides in the double-stranded region.

[0187] In one embodiment, all nucleotides in the sense and antisense strands of an iRNA agent can be modified, including nucleotides that are part of a motif. Each nucleotide can be modified with the same or different modifications, which can include one or more changes to one or both of the non-linked phosphate oxygen and / or one or more of the linking phosphate oxygens; changes to components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; wholesale replacement of the phosphate moiety with a "dephosphorylation" linker; modifications or replacement of natural bases; and replacement or modification of the ribose-phosphate backbone.

[0188] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at repeated positions within the nucleic acid. In some cases, modifications can occur at all of the intended positions in the nucleic acid, but often this is not the case. For example, modifications can occur only at the 3' or 5' terminal positions, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in double-stranded and single-stranded regions, especially at the ends. The 5' or both ends can be phosphorylated.

[0189] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide surrogates in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In certain embodiments, all or some of the bases in the 3' or 5' overhang may be modified, e.g., with the modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, e.g., deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribosugar of the nucleobase, according to modifications known in the art, and modifications of the phosphate group, e.g., phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0190] In one embodiment, each residue in the sense strand and the antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. A strand may contain two or more modifications. In one embodiment, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0191] At least two different modifications are typically present in the sense and antisense strands, and the two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0192] In one embodiment, N a and / or N b includes an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a strand. The alternating nucleotides can refer to one at every other nucleotide or one at every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0193] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0194] In one embodiment, an iRNA agent of the present invention comprises an alternating motif modification pattern in the sense strand that is shifted relative to the alternating motif modification pattern in the antisense strand. This shift can be such that the modification groups of the nucleotides in the sense strand correspond to different modification groups of the nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from 5' to 3' of the strand within the double-stranded region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from 5' to 3' of the strand within the double-stranded region, thereby resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0195] In one embodiment, the iRNA agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the sense strand, which pattern initially has a shift relative to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., 2'-O-methyl modified nucleotides in the sense strand base pair with 2'-F modified nucleotides in the antisense strand, and vice versa. Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification.

[0196] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand interrupts the original modification pattern present in the sense strand and / or antisense strand. This interruption of the modification pattern of the sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand unexpectedly enhances the gene silencing activity against the target gene.

[0197] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different from the modification of the motif. For example, a portion of a sequence containing a motif may be represented by "...N a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications in three consecutive nucleotides, and "N a " and "N b " represents a modification of the nucleotide adjacent to the motif "YYY" that is different from the modification of Y, and N a and N b may be the same or different modifications. a and / or N b may or may not be present if wing modifications are present.

[0198] The iRNA agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present at any nucleotide in the sense strand, the antisense strand, or both strands, at any position in the strand. For example, the internucleotide linkage modification may be present at every nucleotide in the sense strand and / or the antisense strand; each internucleotide linkage modification may be present in an alternating pattern in the sense strand and / or the antisense strand; or the sense strand or the antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications in the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modifications in the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications in the antisense strand.

[0199] In one embodiment, the iRNA comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region may comprise two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between them. The internucleotide bond modification may also be formed to link the overhang nucleotide to a terminal paired nucleotide within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there may be an additional phosphorothioate or methylphosphonate internucleotide bond linking the overhang nucleotide to a paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides can be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0200] In one embodiment, the two nucleotide overhang is at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the iRNA agent can further have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0201] In one embodiment, the double-stranded RNAi agent comprises six to eight phosphorothioate internucleotide linkages, hi one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0202] In one embodiment, the iRNA agent contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., relative to the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With respect to 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, e.g., non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

[0203] In one embodiment, the iRNA agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs in 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 a mismatch pair, e.g., a non-canonical or non-canonical pairing or a pairing including a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0204] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0205] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) (In the formula: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; wherein Nb and Y do not have the same modification; XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides. Preferably, all of YYY are 2'-F modified nucleotides.

[0206] In one embodiment, N a and / or N b contains alternating patterns of modifications.

[0207] In one embodiment, the YYY motif is located at or near the cleavage site of the sense strand. For example, if the iRNA agent has a double-stranded region 17-23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13).

[0208] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand may be represented by the following formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0209] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0210] When the sense strand is represented by formula (Ic), N brepresents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0211] When the sense strand is represented as formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0212] Each of X, Y and Z can be the same or different from each other.

[0213] In other embodiments, i is 0, j is 0, and the sense strand may be represented by the formula: 5'n p -N a -YYY-N a -n q 3'(Ia).

[0214] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0215] In one embodiment, the antisense strand sequence of the iRNA has formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) (In the formula: k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; where N b ' and Y' do not have the same modification; X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides. It can be represented by:

[0216] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0217] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, if the iRNA agent has a double-stranded region 17-23 nucleotides in length, the Y'Y'Y' motif can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13.

[0218] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0219] 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.

[0220] 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).

[0221] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

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

[0223] When the antisense strand is represented by formula (IId), each N bEach N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0224] In other embodiments, k is 0, l is 0, and the antisense strand may be represented by the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia).

[0225] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0226] Each of X', Y' and Z' can be the same or different from each other.

[0227] Each nucleotide in the sense strand and the antisense strand can be independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense strand and the antisense strand can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0228] In one embodiment, the sense strand of the iRNA agent may include a YYY motif at positions 9, 10, and 11 of the strand, counting from the first nucleotide from the 5' end if the double-stranded region is 21 nucleotides; or optionally, counting from the 5' end and starting from the first paired nucleotide in the double-stranded region; Y represents a 2'-F modification. The sense strand may further include a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0229] In one embodiment, the antisense strand may include a Y'Y'Y' motif at positions 11, 12, and 13 of the strand, counting from the first nucleotide from the 5'-end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5'-end; Y' represents a 2'-O-methyl modification. The antisense strand may further include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0230] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0231] Thus, an iRNA agent for use in the methods of the invention can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the iRNA duplex can have the formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; where: Each n may or may not be present p ',n p , n q ', and n q independently represent overhanging nucleotides; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0232] 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; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0233] Exemplary combinations of sense and antisense strands that form iRNA duplexes include the following formulas: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a’ -n q ’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId)

[0234] When an iRNA agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0235] When an iRNA agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence containing, independently, 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0236] When an iRNA agent is represented as formula (IIIc), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0237] When an iRNA agent is represented as formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b ’ Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) can be the same or different from one another.

[0238] When an iRNA agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides, or at least two of the Y nucleotides are base-paired with a corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with a corresponding Y' nucleotide.

[0239] When an iRNA agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can base pair with one of the Z' nucleotides, or at least two of the Z nucleotides base pair with a corresponding Z' nucleotide; or all three of the Z nucleotides base pair with a corresponding Z' nucleotide.

[0240] When an iRNA agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can base pair with one of the X' nucleotides, or at least two of the X nucleotides base pair with a corresponding X' nucleotide; or all three of the X nucleotides base pair with a corresponding X' nucleotide.

[0241] In one embodiment, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, and / or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0242] In one embodiment, when the iRNA agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the iRNA agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In yet another embodiment, when the iRNA agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when an iRNA agent is represented by formula (IIId), N' is attached to the adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker (described below). a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0243] In one embodiment, when the iRNA agent is represented by formula (IIIa), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p'>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0244] In one embodiment, the iRNA agent is a multimer comprising at least two duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0245] In one embodiment, the iRNA agent is a multimer comprising three, four, five, six, or more duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being joined by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0246] In one embodiment, two iRNA agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at one or both of the 5' and 3' ends, and are optionally conjugated to a ligand. Each of the RNAi agents can target the same gene or two different genes; or each of the RNAi agents can target the same gene at two different target sites.

[0247] Various publications describe multimeric iRNA agents that can be used in the methods of the invention, including WO 2007 / 091269, U.S. Patent No. 7,858,769, WO 2010 / 141511, WO 2007 / 117686, WO 2009 / 014887, and WO 2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0248] As described in more detail below, an iRNA agent that includes the conjugation of one or more carbohydrate moieties thereto can optimize one or more properties of the iRNA agent. Often, the carbohydrate moiety is attached to a modified subunit of the iRNA agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been replaced in this manner is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0249] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, containing a functional group, e.g., a hydroxyl group, or generally a backbone, e.g., a phosphate, or a modified phosphate, e.g., sulfur. In certain embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally provide bonds suitable for the incorporation or tethering of another chemical moiety, such as a ligand, to the constituent ring.

[0250] An iRNA agent may be conjugated to a ligand via a carrier, which can be a cyclic group or a cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the cyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0251] In certain embodiments, an iRNA agent for use in a method of the invention is an agent selected from the group of agents listed in any one of Tables 3, 4, 5, 6, and 7. These agents can further comprise a ligand.

[0252] IV. Ligand-Conjugated iRNA Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA.Such moieties include, but are not limited to, cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or lipid moieties such as octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0253] In one embodiment, a ligand alters the distribution, targeting, or lifespan of an iRNA agent into which it is incorporated. In preferred embodiments, a ligand provides improved affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a cell or organ compartment), tissue, organ, or region of the body, e.g., compared to a species lacking such a ligand. Preferred ligands do not participate in pairing of the two strands in a double-stranded nucleic acid.

[0254] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0255] Ligands can also include targeting groups, e.g., cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide, or an RGD peptidomimetic or aptamer.

[0256] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid (cholenic acid), and the like. acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ tetraazamacrocycle conjugate), dinitrophenyl, HRP, or AP.

[0257] A ligand can be a molecule with specific affinity for a protein, e.g., a glycoprotein, or a peptide, e.g., a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, such as a hepatocyte. Ligands can also include hormones and hormone receptors. Ligands can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, or multivalent mannose, multivalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharide, an activator of MAP kinase, or an activator of NF-κB.

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

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

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

[0261] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely made by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may be used in addition or instead. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0262] In the ligand-conjugated oligonucleotides and ligand molecules having sequence-specifically bound nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled in a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0263] When using a nucleotide conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is typically completed before the ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In certain embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizer using phosphoramidites derived from the ligand-nucleoside conjugates in addition to commercially available standard and non-standard phosphoramidites commonly used in oligonucleotide synthesis.

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

[0265] Lipid-based ligands can be used to inhibit, for example, control, the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and 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 conjugates to the kidney.

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

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

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

[0269] B. Cell-penetrating agents In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic and lipophobic phase.

[0270] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, for example, by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0271] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 14)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 15)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 16)) have been shown to be capable of functioning as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., 2004). al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic attached to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is a peptide such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below can be used.

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

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

[0274] C. Carbohydrate conjugates In certain embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo nucleic acid delivery, as described herein, and the compositions are suitable for in vivo therapeutic uses. As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each with an oxygen, nitrogen, or sulfur atom attached to it; or a compound that has as part thereof a carbohydrate moiety composed of one or more monosaccharide units, each of which has at least six carbon atoms (which may be linear, branched, or cyclic), each with an oxygen, nitrogen, or sulfur atom attached to it. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 or greater (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0275] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. [ka] and other N-acetylgalactosamines.

[0276] In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention comprises: [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

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

[0278] In certain embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, including but not limited to, a PK modulator and / or a cell penetrating peptide.

[0279] Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publication Nos. WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0280] D. Linker In certain embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which can be cleavable or non-cleavable.

[0281] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, for example, covalently bonds the two parts of a compound. Linkers are typically a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a group including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkynyl, alkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclyl and alkylaryl, alkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl (wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic); where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0282] A cleavable linking group is one that is sufficiently stable outside a cell, but is cleaved after entering a target cell to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold faster inside the target cell or under first reference conditions (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under second reference conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0283] Cleavable linking groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include oxidizing or reducing enzymes present in cells or reducing agents such as mercaptans that can degrade redox-cleavable linking groups by reduction, which are selective for specific substrates or have no substrate specificity; esterases; agents that can form endosomes or acidic environments, such as agents that cause a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0284] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0285] The linker may contain a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver-targeting ligand may be linked to a cationic lipid via a linker containing an ester group. Because hepatocytes are rich in esterases, this linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

[0286] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synoviocytes.

[0287] In general, the suitability of a candidate cleavable binding group can be evaluated by testing the ability of a degradative agent (or degradative condition) to cleave the candidate binding group. It may also be desirable to test the ability of the candidate cleavable binding group to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between first and second conditions, the first selected to be indicative of cleavage within target cells, and the second selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. This evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 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).

[0288] i. Redox-cleavable linking group In one embodiment, the cleavable linking group is a redox-cleavable linking group that is cleaved after reduction or oxidation. An example of a reductively cleavable linking group is a disulfide bond (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group," or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one may turn to the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which mimic the cleavage rate that can be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In one embodiment, the candidate compound is cleaved at about 10% or less in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster inside cells (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0289] ii. Phosphate-based cleavable linking groups In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group that is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, 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.

[0290] iii. Acid-cleavable linking groups In another embodiment, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less) or by an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can be represented by the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon bonded to the oxygen of the ester (alkoxy group) 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.

[0291] iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by enzymes such as intracellular esterases and amylases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups are represented by the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0292] v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved by enzymes, such as intracellular peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, but do not include all amide functional groups. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0293] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to: [ka] [ka] and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0294] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0295] In one embodiment, the dsRNA of the present invention has the structure represented by formula (XXXII) to (XXXV): [ka] and conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of the following: During the ceremony: each occurrence of q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represents 0 to 20, and the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C each, independently at each occurrence, is absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R"), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5CEach occurrence is independently absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each occurrence is independently a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives can be used with RNAi agents to form a compound of formula (XXXV): [ka] are particularly useful for inhibiting the expression of target genes such as those of In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative.

[0296] Examples of suitable divalent and trivalent branched linking groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas II, VII, XI, X, and XIII.

[0297] Representative patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077 No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779 4,789,737; 4,824,941; 4,835,263; 4,876,335; Specification No. 4,904,582; Specification No. 4,958,013; Specification No. 5,082,830; Specification No. 5,112,963; Specification 5 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,27 Specification No. 2,250; Specification No. 5,292,873; Specification No. 5,317,098; Specification No. 5,371,241, Specification No. 5,391, Specification No. 723; Specification No. 5,416,203, Specification No. 5,451,463; Specification No. 5,510,475; Specification No. 5,512,66 No. 7; No. 5,514,785; No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; No. 5,599,923; No. 5,599,928 and No. 5,688,941; No. 6,294,664; No. 6,320,017; No. 6,576,752; No. 6,783,931; No. 6,900,297;Nos. 7,037,646 and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0298] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the above modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0299] In the context of the present invention, a "chimeric" iRNA compound or "chimera" 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 confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the iRNA for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of iRNA inhibition of gene expression. Consequently, when chimeric dsRNAs are used, comparable results can often be obtained with shorter iRNAs compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can typically be detected by gel electrophoresis and, if desired, by associated nucleic acid hybridization techniques known in the art.

[0300] In some cases, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to improve their activity, cellular distribution, or cellular uptake, and procedures for such conjugation are available in the scientific literature.Such non-ligand moieties include 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, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al. al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative US patents that teach the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the conjugated molecule using an appropriate coupling or activating agent. The conjugation reaction can be carried out with the RNA still attached to a solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0301] V. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, e.g., a cell in a subject, such as a human subject (e.g., a subject in need of an iRNA agent, such as a subject with a disease, disorder, or condition associated with the phosphorylation of fructose by KHK), can be achieved in several different ways. For example, delivery can be achieved by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery can also be achieved directly by administering a composition containing an iRNA, e.g., a dsRNA, to the subject. Alternatively, in vivo delivery can be achieved indirectly by administering one or more vectors that encode and direct the expression of the iRNA. Examples of these alternatives are described further below.

[0302] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian R.L., (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider when delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, e.g., by direct injection or implantation into the tissue, or by administering the formulation locally. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be adversely affected by or degrade the agent, and can reduce the total dose of the iRNA molecule administered. Several studies have demonstrated successful knockdown of gene products when iRNAs are administered locally. For example, intraocular delivery of VEGF dsRNA via intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al., (2003) Mol. Vis. 9:210-216) have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice can reduce tumor volume (Pille, J. et al., (2005) Mol. Ther. 11:267-274) and prolong the survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523).RNA interference can be delivered locally to the central nervous system by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P.H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G.T. et al. (2004) Neuroscience 129:521-528; Thakker, E.R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602) and locally to the lung by intranasal administration (Howard, K.A. et al. (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55) have also demonstrated success. To administer iRNA systemically for disease treatment, the RNA can be modified or delivered using a drug delivery system; both methods serve to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutical carrier can also enable targeting of iRNA compositions to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to improve cellular uptake and prevent degradation. For example, iRNAs against ApoB conjugated to lipophilic cholesterol moieties were administered systemically to mice, resulting in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, J. et al., (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNAs can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate binding of iRNA molecules (which are negatively charged) and also improve interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNAs or induced to form vesicles or micelles that encapsulate iRNAs (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNAs when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N. et al. (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S. et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y. et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E. et al. (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, iRNAs are complexed with cyclodextrins for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

[0303] A. Vector-encoded iRNA of the invention iRNAs targeting the C5 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A., et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00 / 22113; Conrad, International PCT Publication No. WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a few hours to a few weeks) or sustained (from weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0304] The individual strands of the iRNA can be transcribed from a promoter in an expression vector. Two separate expression vectors can be co-introduced into a target cell (e.g., by transfection or infection), where two separate strands are expressed to produce, for example, dsRNA. Alternatively, each individual strand of the dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence to form a stem-loop structure.

[0305] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing the iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic expression vectors are well known in the art and are available from numerous commercial sources. Such vectors are typically provided containing convenient restriction sites for inserting the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells transplanted from the patient and then reintroduced into the patient, or by any other means that allows for introduction into the desired target cells.

[0306] iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of one week or more are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated using a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using a marker that confers resistance to certain environmental factors (e.g., antibiotics and drugs) on the transfected cells, such as hygromycin B resistance.

[0307] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors, or avian pox, e.g., canarypox or fowlpox, poxvirus vectors; and (j) helper-dependent or attenuated adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may optionally include viral sequences for transfection. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are discussed further below.

[0308] Vectors useful for delivery of iRNA will contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulatable / inducible expression.

[0309] Expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as blood glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0310] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding the iRNAs are cloned into one or more vectors, which facilitate delivery of the nucleic acid to a patient. Further details about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references demonstrating the use of retroviral vectors in gene therapy are Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0311] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155; Mastrangeli et al. (1992), J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). AV vectors suitable for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0312] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNAs can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101; Fisher KJ et al. (1996), J.Virol, 70:520-532; Samulski R et al. (1989), J.Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0313] Another viral vector suitable for delivery of the iRNA of the invention is a poxvirus, such as a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or an avian pox, such as fowlpox or canarypox.

[0314] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by engineering the vector to express different capsid protein serotypes. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0315] The pharmaceutical preparation of the vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0316] VI. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier is also provided herein. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with KHK gene expression or activity. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral administration, e.g., subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, e.g., by injection into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the present invention can be administered at a dosage sufficient to inhibit KHK gene expression.

[0317] The pharmaceutical composition may be administered by intravenous infusion over a period of time such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24 minutes, or about 25 minutes. Administration may be repeated periodically, for example, weekly or biweekly (i.e., every two weeks) for one month, two months, three months, four months, or more. After the initial treatment regimen, the therapeutic agent may be administered less frequently. For example, after three months of weekly or biweekly administration, administration may be repeated once a month for six months, one year, or more.

[0318] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more subdoses at appropriate intervals throughout the day, or even via continuous infusion or delivery via sustained-release formulations. In such cases, each subdose must contain a correspondingly smaller amount of iRNA to achieve the total daily dosage. The dosage unit can also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of the iRNA over a period of several days. Sustained-release formulations are well known in the art and can be used with the agents of the invention, as they are particularly useful for delivering agents to specific sites. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0319] In other embodiments, a single dose of the pharmaceutical composition can be administered prolonged, with subsequent doses administered at intervals of no more than 3, 4, or 5 days, or no more than 1, 2, 3, or 4 weeks. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered weekly. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered bi-monthly.

[0320] Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can influence the dosage and duration required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for individual iRNAs encompassed by the invention can be estimated using conventional methodology or based on in vivo testing using appropriate animal models as described elsewhere herein.

[0321] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary administration, e.g., by inhalation or insufflation of powders or aerosols, such as with a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous administration, e.g., via an implanted device; or intracranial administration, e.g., via intraparenchymal, intrathecal, or intraventricular administration.

[0322] The iRNA can be delivered to target a specific tissue, such as the liver (e.g., hepatocytes of the liver).

[0323] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is mixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleyltetramethylaminopropyl DOTAP and dioleylphosphatidylethanolamine DOTMA). The iRNAs featured in the present invention can be encapsulated in liposomes or complexed to liposomes, particularly cationic liposomes. Alternatively, the iRNAs can be complexed to lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

[0324] A. iRNA formulations containing membrane molecular assemblies iRNAs for use in the compositions and methods of the present invention can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar and multilamellar vesicles, whose membrane is formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior and typically does not contain the iRNA composition, although in some cases it may. Liposomes are useful for transporting and delivering active ingredients to sites of action. Because the liposome membrane is structurally similar to biological membranes, when the liposome is attached to a tissue, the liposome bilayer fuses with the cell membrane bilayer. As the liposome and cell fuse, the aqueous contents, including the iRNA, are delivered to the cell, where the iRNA can specifically bind to the target RNA and mediate iRNA activity. In some cases, the liposomes are also specifically targeted, for example, to direct the iRNA to a particular cell type.

[0325] Liposomes containing iRNA agents can be prepared by a variety of methods. In one example, the lipid components of the liposome are dissolved in a detergent so that micelles are formed with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. A preparation of the iRNA agent is then added to the micelles containing the lipid components. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form a liposome. After condensation, the detergent is removed, for example, by dialysis, to yield a liposomal formulation of the iRNA agent.

[0326] If necessary, a carrier compound that aids in condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to aid in condensation.

[0327] Methods for producing stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., M. Mol. 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. al., Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw and extrusion (see, e.g., Mayer et al., Biochim. Biophys. Acta 858:161, 1986). When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging preparations of iRNA agents into liposomes.

[0328] Liposomes are divided into two major 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 transported into endosomes. The acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0329] pH-sensitive, negatively charged liposomes entrap nucleic acids rather than complexing them. Because both the nucleic acid and the lipid are similarly charged, repulsion occurs rather than complexation. Nevertheless, some nucleic acids are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0330] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic membrane-fusogenic liposomes are primarily formed from dioleylphosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0331] Other exemplary methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss, EMBO J. 11:417, 1992.

[0332] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. The results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A in different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4(6)466).

[0333] Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) those that are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that the enhanced circulation half-life of these sterically stabilized liposomes, at least for those containing gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0334] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported the use of monosialoganglioside G, which improves the blood half-life of liposomes. M1 reported the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. These findings are detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0335] In one embodiment, cationic liposomes are used.Cationic liposomes have the advantage that they can fuse with cell membranes.Non-cationic liposomes cannot fuse with cell membranes as efficiently, but they can be taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

[0336] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; and liposomes can protect iRNA agents encapsulated in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger, and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0337] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids, forming lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the plasma membrane of tissue culture cells, resulting in delivery of iRNA agents (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0338] DOTMA analogue, 1,2-bis(oleyloxy)-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 into living tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously adhere to negatively charged cell surfaces, fuse with the cell membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is attached by an ester rather than an ether bond.

[0339] Other reported cationic lipid compounds include those conjugated to one of two types of lipids and conjugated to various moieties, including, for example, carboxyspermine, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

[0340] Another cationic lipid conjugate involves derivatizing lipids with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, prepared by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0341] Liposome formulations are particularly suitable for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug in the desired target, and the ability to administer iRNA agents to the skin. In some implementations, liposomes are used to deliver iRNA agents to epidermal cells and to promote the penetration of iRNA agents into dermal tissues, such as the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been reported (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992:259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al., Gene 56:267-276, 1987; Nicolau, C. et al. (1987) Meth. Enz. 149:157-176, 1987; Straubinger, R M and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0342] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0343] Liposomes containing iRNA can be made highly deformable. Such deformability can allow the liposomes to pass through pores smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be made by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing iRNA agents can be delivered, for example, by subcutaneous injection, to deliver iRNA agents to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to their lipid properties, these transferosomes can be self-optimizing (e.g., adaptable to the shape of pores), self-repairing, often reaching their target without fracture, and often self-loading.

[0344] Other formulations suitable for the present invention are described in U.S. Provisional Patent Applications Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations suitable for the present invention.

[0345] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can also be described as lipid droplets, which, because of their high deformability, can easily penetrate pores smaller than the droplets. Transfersomes can adapt to the environment in which they are used, for example, they are self-optimizing (adapting to the shape of pores in the skin), self-repairing, often reach their targets without fragmentation, and are often self-loading. 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 solution containing serum albumin.

[0346] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of categorizing the different surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0347] If the surfactant molecule is not ionized, the surfactant is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0348] When surfactant molecule dissolves or disperses in water, if it carries negative charge, this surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

[0349] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic.Cationic surfactants include quaternary ammonium salts and ethoxylated amines.Quaternary ammonium salts are the most commonly used members of this class.

[0350] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0351] The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0352] The iRNA for use in the method of the present invention can also be provided as a micelle formulation. " Micelle " is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure, with all hydrophobic parts of the molecule facing inward, and the hydrophilic parts remaining in contact with the surrounding aqueous phase. When the environment is hydrophobic, the opposite arrangement exists.

[0353] Mixed micelle formulations suitable for transdermal delivery contain an aqueous solution of the siRNA composition, alkali metal C8-C 22 They can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compounds may be added simultaneously with or after the addition of alkali metal alkyl sulfates. Mixed micelles can be formed by virtually any type of mixing of components, but more vigorous mixing is preferred to provide smaller micelles.

[0354] In one method, a first micelle composition containing an siRNA composition and at least an alkali metal alkyl sulfate is prepared. 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 the siRNA composition, the alkali metal alkyl sulfate, and at least one of the micelle-forming compounds, followed by adding the remaining micelle-forming compounds while vigorously mixing.

[0355] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0356] To deliver a micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, which is then filled with a propellant. The propellant, under pressure, is in liquid form in the dispenser. The ratio of the components is adjusted so that there is one aqueous phase and one propellant phase, i.e., one phase. If two phases are present, the dispenser must be shaken before dispensing a portion of its contents, for example, via a metered valve. The medicinal dose is then expelled from the metered valve in the form of a fine spray.

[0357] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0358] The specific concentrations of the essential components can be determined by relatively simple experimentation. For absorption via the oral cavity, it is often desirable to increase the dosage, for example, by at least two or three times, that for injection or administration via the gastrointestinal tract.

[0359] B. Lipid particles The iRNA, ie, dsRNA, of the present invention may be fully encapsulated in a lipid formulation, such as an LNP, or may form other nucleic acid-lipid particles.

[0360] The term "LNP" as used herein refers to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs have an extended circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them extremely useful for systemic applications. LNPs include "pSPLPs," which contain encapsulated condensing agent-nucleic acid complexes, as described in PCT Publication No. WO 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Additionally, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and PCT Publication No. WO 96 / 40964.

[0361] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above ranges are also considered part of the invention.

[0362] Examples of cationic lipids include 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-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-di Methylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof , (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0363] 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.

[0364] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mol percent), and have a particle size of 63.0±20 nm and a 0.027 siRNA / lipid ratio.

[0365] Ionic / non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N The lipid may be an anionic or neutral lipid, including 1-(2-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid, when cholesterol is included, may comprise about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0366] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol%, or 2 mol% of the total lipid present in the particle.

[0367] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0368] In one embodiment, lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Respective stock solutions in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-Ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., in sodium acetate (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 usually form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder such as the Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate-buffered saline (PBS), for example, at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. [ka]

[0369] LNP01 formulations are described, for example, in International Application Publication No. WO 2008 / 042973, which is incorporated herein by reference.

[0370] Further exemplary lipid-dsRNA formulations are described in Table 1.

[0371] [Table 1]

[0372] [Table 2]

[0373] DSPC: Distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000) Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminepropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0374] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application Nos. 61 / 148,366, filed January 29, 2009; 61 / 156,851, filed March 2, 2009; 61 / 156,851, filed June 10, 2009; 61 / 228,373, filed July 24, 2009; 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0375] Formulations containing MC3 are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference.

[0376] Formulations containing ALNY-100 are described, for example, in International Patent Application No. PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0377] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0378] Synthesis of ionic / cationic lipids Any of the compounds, e.g., cationic lipids, used in the nucleic acid-lipid particles of the present invention can be prepared by known organic synthesis techniques, including those methods described in more detail in the Examples. All substituents are as defined below unless otherwise indicated.

[0379] "Alkyl" means a straight-chain or branched-chain, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched-chain alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0380] "Alkenyl" refers to an alkyl as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight and branched chain alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0381] "Alkynyl" means any alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative straight and branched chain alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0382] "Acyl" means any alkyl, alkenyl, or alkynyl group in which the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0383] "Heterocycle" means a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocycle that is either saturated, unsaturated, or aromatic and contains one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be bonded via any heteroatom or carbon atom. Heterocycle includes heteroaryl, as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0384] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle," when substituted, mean that at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, -CN, -ORx, -NRxRy, -NRxC(=O)Ry, -NRxS02Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy, where n is 0, 1 or 2 and Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORx, heterocycle, -NRxRy, -NRxC(=O)Ry, -NRxS02Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy.

[0385] "Halogen" means fluoro, chloro, bromo and iodo.

[0386] In certain embodiments, the methods of the present invention may require the use of protecting groups. Protecting group methods are well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, TW et al., Wiley-Interscience, New York City, 1999). Briefly, in the context of the present invention, a protecting group is any group that reduces or eliminates the undesired reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during a specific reaction and then removed to reveal the original functional group. In certain embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the undesired reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0387] Synthesis of Formula A In one embodiment, the nucleic acid-lipid particles of the present invention have Formula A: [ka] In some embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Generally, the lipid of formula A above can be prepared by the following reaction scheme 1 or 2, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 can be taken together to form an optionally substituted heterocycle.

[0388] [ka] Lipids A (wherein R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be optionally substituted; R3 and R4 are independently lower alkyl; or R3 and R4 can be joined to form an optionally substituted heterocycle) can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 provides ketal 3. Treatment of ketal 3 with amine 4 provides lipids of formula A. Lipids of formula A can be converted to the corresponding ammonium salt using an organic salt of formula 5 (wherein X is an anionic counterion selected from halogen, hydroxide, phosphate, sulfate, etc.).

[0389] [ka] Alternatively, the starting material ketone 1 can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 6 and 7 gives ketone 1. Conversion of ketone 1 to the corresponding lipid of formula A is depicted in Scheme 1.

[0390] Synthesis of MC3 DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was prepared as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid and then with dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed on a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent was removed to give a colorless oil (0.54 g).

[0391] Synthesis of ALNY-100 The synthesis of ketal 519 [ALNY-100] was carried out according to Scheme 3. [ka]

[0392] Synthesis of 515 To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mL of anhydrous THF in a two-necked round-bottom flask (1 L) was slowly added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF at 0°C under a nitrogen atmosphere. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. The progress of the reaction was monitored by TLC. After completion of the reaction (by TLC), the mixture was cooled to 0°C and quenched by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 h and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. The volatiles were removed under reduced pressure to give the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1 H-NMR (DMSO, 400MHz): δ = 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66~2.60 (m, 2H), 2.50~2.45 (m, 5H).

[0393] Synthesis of 516 To a stirred solution of compound 515 in 100 mL of dry DCM in a 250 mL two-neck round-bottom flask, NEt3 (37.2 mL, 0.2669 mol) was added and cooled to 0 °C under a nitrogen atmosphere. N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM was slowly added, and the reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC), the mixture was washed successively with 1 N HCl solution (1 × 100 mL) and saturated NaHCO3 solution (1 × 50 mL). The organic layer was then dried over anhydrous Na2SO4, and the solvent was evaporated to give crude material, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400MHz): δ=7.36~7.27(m,5H), 5.69(s,2H), 5.12(s,2H), 4.96(br.,1H)2.74(s,3H), 2.60(m,2H), 2.30~2.25(m,2H). LC-MS[M+H]-232.3(96.94%).

[0394] Synthesis of 517A and 517B Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of a 10:1 solution of acetone and water in a 500 mL single-neck round-bottom flask, and N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added thereto, followed by 4.2 mL of a 7.6% solution of OsO (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (approximately 3 h), the mixture was quenched by the addition of solid NaSO, and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over anhydrous NaSO, and the solvent was removed under reduced pressure. Silica gel column chromatography purification of the crude material afforded a mixture of diastereomers, which were separated by preparative HPLC. Yield: 6 g of crude 517A-Peak-1 (white solid), 5.13 g (96%). H-NMR (DMSO, 400 MHz): δ = 7.39–7.31 (m, 5H), 5.04 (s, 2H), 4.78–4.73 (m, 1H), 4.48–4.47 (d, 2H), 3.94–3.93 (m, 2H), 2.71 (s, 3H), 1.72–1.67 (m, 4H). LC-MS: [M+H] = 266.3, [M+NH] = 283.5 present, HPLC = 97.86%. Stereochemistry confirmed by X-ray.

[0395] Synthesis of 518 Using a procedure similar to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. H-NMR (CDCl, 400 MHz): δ = 7.35–7.33 (m, 4H), 7.30–7.27 (m, 1H), 5.37–5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58–4.57 (m, 2H), 2.78–2.74 (m, 7H), 2.06–2.00 (m, 8H), 1.96–1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37–1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

[0396] General procedure for the synthesis of compound 519 A solution of compound 518 (1 equiv.) in hexane (15 mL) was added dropwise to an ice-cooled solution of LAH in THF (1 M, 2 equiv.). After complete addition, the mixture was heated at 40° C. for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous NaSO, then filtered through Celite and reduced to an oil. Column chromatography afforded pure 519 (1.3 g, 68%), which was obtained as a colorless oil. 13 C NMR δ = 130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; electrospray MS (+ve): C 44 H 80 Molecular weight (M+H) for NO2 calculated 654.6, found 654.6.

[0397] Formulations prepared by either standard or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. The formulation should be a whitish, translucent solution without aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be approximately 20-300 nm in size, e.g., 40-100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation and the entrapped fraction can be estimated using a dye exclusion assay. A sample of the formulated dsRNA may be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by comparing the signal from the sample containing the surfactant against a standard curve. The entrapped fraction is determined by subtracting the "free" dsRNA content (measured by the signal in the absence of surfactant) from the total dsRNA content. The percent of entrapped dsRNA is typically >85%. For SNALP formulations, particle sizes are at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Suitable ranges are typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0398] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be desirable. In some embodiments, oral formulations are those in which the dsRNA characterizing the present invention is administered with one or more permeation enhancers, surfactants, and chelating agents. Suitable surfactants include fatty acids and / or esters or their salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic 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-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as a fatty acid / salt in combination with a bile acid / salt. An exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA characterizing the present invention can be orally delivered in the form of granules, including spray-dried particles, or complexed to form micro- or nanoparticles.dsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkylcyanoacrylates, DEAE-derivatized polyimines, pullulan, 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(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate. Oral formulations for dsRNA and their formulations are described in U.S. Pat. No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0399] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0400] The pharmaceutical compositions of the present invention include, but are not limited to, liquids, emulsions, and liposome-containing formulations. These compositions can be made from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases such as hepatocarcinoma, liver-targeting formulations are particularly preferred.

[0401] The pharmaceutical formulations of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with pharmaceutical carriers or excipients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0402] 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, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer.

[0403] C. Further Formulations i. Emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets, usually greater than 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, in 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., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w) types. When an aqueous phase is finely divided and dispersed as minute droplets into a bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when an oil phase is finely divided and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions can contain additional components in addition to the dispersed phase and active drug, which may be present in the aqueous phase, as a solution in the oil phase, or as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions can be multiple emulsions consisting of three or more phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages not offered by simple binary emulsions. Multiple emulsions in which individual oil droplets of an o / w emulsion surround small water droplets constitute w / o / w emulsions. Similarly, a system of oil droplets surrounded by globules of water stabilized in a continuous oil phase provides an o / w / o emulsion.

[0404] Emulsions are characterized by having little or no thermodynamic stability.In many cases, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase and is maintained in this form through the use of emulsifiers or the viscosity of the formulation.Either phase of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams.Other means of stabilizing emulsions include the use of emulsifiers, which can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0405] Synthetic surfactants, also known as surface active agents, have found widespread applicability in the formulation of emulsions and have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199). Surfactants are usually amphiphilic, comprising a hydrophilic portion and a hydrophobic portion. The ratio of hydrophilicity to hydrophobicity of surfactant is called hydrophilic / lipophilic balance (HLB), which is a valuable tool for classifying and selecting surfactants when preparing formulations.Surfactants can be classified into different types based on the nature of hydrophilic group, namely 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, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285).

[0406] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases, such as anhydrous lanolin and hydrophilic petrolatum, possess hydrophilic properties that allow them to incorporate water to form water-in-oil emulsions while still maintaining their semisolid consistency. Finely divided solids have been used as good emulsifiers, especially in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0407] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions, including fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0408] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around droplets of the dispersed phase and by increasing the viscosity of the external phase.

[0409] Emulsions often contain many ingredients, such as carbohydrates, proteins, sterols, and phosphatides, which can easily support the growth of microorganisms, so these preparations often incorporate preservatives. Commonly used preservatives in preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also usually added to emulsion preparations to prevent the preparation from deteriorating. The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0410] The application of emulsion formulations via the dermal, oral and parenteral routes and their manufacturing methods are reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of formulation and effectiveness in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional formulations are among the materials commonly administered orally as oil-in-water emulsions.

[0411] ii. Microemulsions In one embodiment of the present invention, iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphile that is a single, optically isotropic, and thermodynamically stable liquid solution (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution, followed by the addition of a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a clear system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared using a combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in: Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0412] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with extensive knowledge of how to formulate 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, in 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 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 in a formulation of thermodynamically stable droplets that form spontaneously.

[0413] Surfactants used in preparing microemulsions, alone or in combination with cosurfactants, include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750). The cosurfactant, typically a short-chain alcohol such as ethanol, 1-propanol, or 1-butanol, serves to increase interfacial fluidity by penetrating the surfactant film, resulting in the formation of an irregular film due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include materials such as, but not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0414] Microemulsions are particularly interesting from the standpoint of drug solubilization and drug absorption enhancement. Lipid-based microemulsions (both o / w and w / o) have been proposed to improve the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105, 7,063,860, 7,070,802, 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105, 7,063,860, 7,070,802, 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can form spontaneously when the components of the microemulsion are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or iRNA. Microemulsions are also effective for the efficient delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improved local cellular uptake of iRNA and nucleic acids.

[0415] The microemulsions of the present invention may also contain additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve formulation properties and enhance absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0416] iii. Particulates The iRNA agents of the invention may be incorporated into particles, e.g., microparticles. Microparticles can be produced by spray drying, but may also be produced by other methods, including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0417] iv. Penetration enhancers In one embodiment, the present invention utilizes various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to the skin of animals. Most drugs exist in solution in both ionized and non-ionized forms. However, typically, only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the permeability of lipophilic drugs.

[0418] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above types of penetration enhancers is described in more detail below.

[0419] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of iRNA through mucosal membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0420] Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1~20 Included are alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their monoglycerides and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, for example, Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0421] The physiological role of bile includes facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee, et al.,Critical Reviews in Therapeutic Drug Carrier Systems,1991,page 92;Swinyard,Chapter 39 In:Remington's Pharmaceutical Sciences,18th Ed.,Gennaro,ed.,Mack Publishing Co.,Easton,Pa.,1990,pp.782-783;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0422] Chelators used in connection with the present invention can be defined as compounds that remove metal ions from solution by forming complexes with the metal ions, resulting in enhanced absorption of iRNA through mucosal membranes. With regard to the use of chelators as penetration enhancers in the present invention, chelators have the added advantage of also acting as DNase inhibitors, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelators (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives (enamines) of β-diketones (see, for example, Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0423] As used herein, non-chelating, non-surfactant penetration enhancers can be defined as compounds that exhibit minimal activity as chelators or surfactants but nonetheless enhance the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of this type of penetration enhancer include unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacycloalkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92), and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0424] Agents that enhance cellular uptake of iRNA can also be added to pharmaceutical and other compositions of the present invention. For example, cationic lipids such as lipofectin (Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., PCT Application WO 97 / 30731) are known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), iRNAMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;(Madison, WI), TransFast (trademark) Transfection Reagent (Promega; Madison, WI), Tfx (trademark)-20 Reagent (Promega; Madison, WI), Tfx (trademark)-50 Reagent (Promega; Madison, WI), DreamFect (trademark) (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass; aD1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM) / LipoGen(TM) (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER(TM) transfection Reagent (Genlantis; San Examples of suitable fusion proteins include RiboFect (Bioline; Taunton, MA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), and HiFect™ (B-Bridge International, Mountain View, CA, USA).

[0425] Other agents can be used to enhance penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azone, and terpenes such as limonene and menthone.

[0426] v. Carrier Certain compositions of the present invention also incorporate a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or analog thereof that may be inert (i.e., not possessing biological activity) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or promoting its removal from the circulation. Co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter, can substantially reduce the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, possibly due to competition between the carrier compound and the nucleic acid for a common receptor. For example, recovery of partial phosphorothioates in liver tissue can be reduced when they are co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0427] vi. excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected to provide the desired bulk, consistency, etc. when combined with the nucleic acid and other desired components of the pharmaceutical composition, taking into account the planned method of administration. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); tablet disintegrants (such as starch, sodium starch glycolate, and the like); and wetting agents (such as sodium lauryl sulfate, and the like).

[0428] Suitable organic or inorganic excipients that are pharmaceutically acceptable for parenteral administration and do not adversely react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0429] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions, or solutions of nucleic acids in liquid or solid oil bases in common solvents such as alcohol. Solutions can also contain buffers, diluents, and other suitable additives. Suitable organic or inorganic excipients that are pharmaceutically acceptable for parenteral administration and do not adversely react with nucleic acids can be used.

[0430] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0431] vii. Other components The compositions of the present invention may also contain other auxiliary components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically active ingredients, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, these materials should not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if desired, mixed with auxiliary substances that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0432] The aqueous suspension may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer.

[0433] In some embodiments, pharmaceutical compositio...

Claims

1. A double-stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by no more than 3 nucleotides.

2. 1. A double-stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), comprising a sense strand and an antisense strand which form a double-stranded region, wherein the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 3, 4, 5, 6, and 7.

3. 3. The dsRNA of claim 2, wherein the sense and antisense strands comprise a sequence selected from the group consisting of AD-63851, AD-63820, AD-63853, AD-63839, AD-63854, AD-63855, and AD-63886, and any one of the sequences disclosed in any one of Tables 3, 4, 5, 6, and 7.

4. The double-stranded RNAi agent of any one of claims 1 to 3, comprising at least one modified nucleotide.

5. The modified nucleotide may be a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a terminal nucleotide attached to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, a non-basic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified 5. The dsRNA of claim 4, wherein the nucleotide is selected from the group consisting of nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics.

6. 6. The dsRNA of claim 5, wherein at least one of the modified nucleotides is selected from the group consisting of 2'-O-methyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

7. A double-stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by no more than 3 nucleotides; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; A double-stranded RNAi agent, wherein the sense strand is conjugated to a ligand attached at the 3' end.

8. 8. The double-stranded RNAi agent of claim 7, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

9. The double-stranded RNAi agent of claim 7, wherein the sense strand and the antisense strand comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ from any one of the antisense sequences listed in any one of Tables 3, 4, and 5 by no more than 3 nucleotides.

10. The modified nucleotide is a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a terminal nucleotide attached to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, a non-basic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide.

10. The double stranded RNAi agent of any one of claims 7 to 9, wherein the nucleotide is selected from the group consisting of: 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, and nucleotides comprising 5'-phosphate mimetics.

11. 10. The double stranded RNAi agent of claim 1, 2 or 7, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

12. 10. The double-stranded RNAi agent of claim 1, 2, or 7, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

13. The double-stranded RNAi agent of claim 1 or 2, further comprising a ligand.

14. A pharmaceutical composition comprising the double-stranded RNAi agent of claim 1 or 2 and a lipid formulation.

15. 15. The pharmaceutical composition of claim 14, wherein the lipid formulation comprises a lipid nanoparticle (LNP).

16. 16. The pharmaceutical composition of claim 15, wherein the lipid nanoparticle (LNP) comprises MC3 lipid.

17. 1. A composition comprising a modified antisense polynucleotide agent, the agent being capable of inhibiting expression of ketohexokinase (KHK) in a cell and comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 3, 4, 5, 6, and 7, wherein the polynucleotide is from about 14 to about 30 nucleotides in length.

18. A double-stranded RNAi agent capable of inhibiting expression of ketohexokinase (KHK) in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having the formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; Each n may be present or absent. ｐ , n ｐ ', n ｑ , and n ｑ ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; N ｂ the modification on Y is different from the modification on N ｂ the modification on Y' is different from the modification on Y'; and the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

19. 20. The double-stranded RNAi agent of claim 18, wherein i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1.

20. 20. The double-stranded RNAi agent of claim 18, wherein k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1.

21. 20. The double-stranded RNAi agent of claim 18, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

22. 20. The double-stranded RNAi agent of claim 18, wherein the YYY motif is at or near the site of cleavage in the sense strand.

23. 20. The double-stranded RNAi agent of claim 18, wherein the Y'Y'Y' motif is present at positions 11, 12, and 13 of the antisense strand at the 5' end.

24. The double-stranded RNAi agent of claim 23, wherein said Y' is 2'-O-methyl.

25. Formula (III) is represented by formula (IIIa): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -Y'Y'Y'-N ａ’ -n ｑ’ 5'(IIIa) The double-stranded RNAi agent of claim 18 , wherein the double-stranded RNAi agent is represented by:

26. Formula (III) is a compound represented by formula (IIIb): Sense: 5'n ｐ -N ａ -YYY-N ｂ -ZZZ-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -Y'Y'Y'-N ｂ’ -Z'Z'Z'-N ａ’ -n ｑ’ 5'(IIIb) (In the formula, each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides. The double-stranded RNAi agent of claim 18 , wherein the double-stranded RNAi agent is represented by:

27. Formula (III) represents the formula (IIIc): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -X'X'X'-N ｂ’ -Y'Y'Y'-N ａ’ -n ｑ’ 5'(IIIc) (In the formula, each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides. The double-stranded RNAi agent of claim 18 , wherein the double-stranded RNAi agent is represented by:

28. Formula (III) represents the formula (IIId): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ｂ -ZZZ-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -X'X'X'-N ｂ’ -Y'Y'Y'-N ｂ’ -Z'Z'Z'-N ａ’ -n ｑ’ 5'(IIId) (In the formula, each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides. The double-stranded RNAi agent of claim 18 , wherein the double-stranded RNAi agent is represented by:

29. 20. The double-stranded RNAi agent of any one of claims 1, 2, 7, or 18, wherein the double-stranded region is 15-30 nucleotide pairs in length.

30. 30. The double-stranded RNAi agent of claim 29, wherein the double-stranded region is 17-23 nucleotide pairs in length.

31. 30. The double-stranded RNAi agent of claim 29, wherein the double-stranded region is 17-25 nucleotide pairs in length.

32. 30. The double-stranded RNAi agent of claim 29, wherein the double-stranded region is 23-27 nucleotide pairs in length.

33. 30. The double-stranded RNAi agent of claim 29, wherein the double-stranded region is 19-21 nucleotide pairs in length.

34. 30. The double-stranded RNAi agent of claim 29, wherein the double-stranded region is 19-23 nucleotide pairs in length.

35. 30. The double-stranded RNAi agent of claim 29, wherein the double-stranded region is 21-23 nucleotide pairs in length.

36. The double stranded RNAi agent of any one of claims 1, 2, 7 or 18, wherein each strand has 15-30 nucleotides.

37. The double stranded RNAi agent of any one of claims 1, 2, 7 or 18, wherein each strand has 19-30 nucleotides.

38. 19. The double stranded RNAi agent of claim 18, wherein the modification on the nucleotide is selected from the group consisting of LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

39. 39. The double-stranded RNAi agent of claim 38, wherein the modification on the nucleotide is a 2'-O-methyl or a 2'-fluoro modification.

40. 20. The double-stranded RNAi agent of any one of claims 7, 13, or 18, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

41. The ligand is 【Chemistry 1】 20. The double-stranded RNAi agent of claim 7, 13, or 18, wherein

42. 20. The double-stranded RNAi agent of any one of claims 7, 13, or 18, wherein the ligand is attached to the 3' end of the sense strand.

43. Schematic diagram below 【Chemistry 2】 wherein X is O or S.

44. 20. The double-stranded RNAi agent of any one of claims 1, 2, 7, or 18, further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

45. 45. The double stranded RNAi agent of claim 44, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand.

46. 46. ​​The double stranded RNAi agent of claim 45, wherein said strand is the antisense strand.

47. 46. ​​The double stranded RNAi agent of claim 45, wherein said strand is the sense strand.

48. 45. The double stranded RNAi agent of claim 44, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand.

49. 49. The double stranded RNAi agent of Claim 48, wherein said strand is the antisense strand.

50. 49. The double stranded RNAi agent of Claim 48, wherein said strand is the sense strand.

51. 45. The double stranded RNAi agent of claim 44, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand.

52. 52. The double stranded RNAi agent of Claim 51, wherein said strand is the antisense strand.

53. The double-stranded RNAi agent of claim 44, wherein the RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages.

54. 54. The double-stranded RNAi of claim 53, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

55. 20. The double-stranded RNAi agent of any one of claims 1, 2, 7, or 18, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

56. 20. The double-stranded RNAi agent of claim 18, wherein said Y nucleotide comprises a 2'-fluoro modification.

57. 20. The double-stranded RNAi agent of claim 18, wherein said Y' nucleotide comprises a 2'-O-methyl modification.

58. 20. The double-stranded RNAi agent of claim 18, wherein p'>0.

59. 20. The double-stranded RNAi agent of claim 18, wherein p'=2.

60. 60. The double-stranded RNAi agent of claim 59, wherein q'=0, p=0, q=0, and the p' overhanging nucleotide is complementary to the target mRNA.

61. 60. The double-stranded RNAi agent of claim 59, wherein q'=0, p=0, q=0, and the p' overhanging nucleotide is non-complementary to the target mRNA.

62. 60. The double-stranded RNAi agent of claim 59, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

63. At least one n ｐ 63. The double stranded RNAi agent of any one of claims 58-62, wherein: ' is linked to an adjacent nucleotide via a phosphorothioate bond.

64. All n ｐ 64. The double stranded RNAi agent of claim 63, wherein said nucleotide is linked to an adjacent nucleotide via a phosphorothioate bond.

65. 20. The double-stranded RNAi agent of claim 1, 2, 7, or 18, selected from the group of RNAi agents listed in any one of Tables 3, 4, 5, 6, and 7.

66. 20. The double stranded RNAi agent of claim 7 or 18, wherein the RNAi agent is selected from the group of AD-63851, AD-63820, AD-63853, AD-63839, AD-63854, AD-63855, and AD-63886.

67. A double-stranded RNAi agent for inhibiting expression of ketohexokinase (KHK), comprising: comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprises at least 15 contiguous nucleotides which differ from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides which differ from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by no more than 3 nucleotides; substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end; A double-stranded RNAi agent, wherein the sense strand is conjugated at its 3' end to one or more GalNAc derivatives attached via a branched divalent or trivalent linker.

68. 68. The double-stranded RNAi agent of claim 67, wherein all of said nucleotides of said sense strand and all of said nucleotides of said antisense strand comprise a modification.

69. 1. A double-stranded RNAi agent capable of inhibiting expression of ketohexokinase (KHK) in a cell, the agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having the formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; Each n may be present or absent. ｐ , n ｐ ', n ｑ , and n ｑ ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

70. 1. A double-stranded RNAi agent capable of inhibiting expression of ketohexokinase (KHK) in a cell, the agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having the formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

71. 1. A double-stranded RNAi agent capable of inhibiting expression of ketohexokinase (KHK) in a cell, the agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having the formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; A double-stranded RNAi agent represented by:

72. 1. A double-stranded RNAi agent capable of inhibiting expression of ketohexokinase (KHK) in a cell, the agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having the formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand comprises at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; A double-stranded RNAi agent represented by:

73. 1. A double-stranded RNAi agent capable of inhibiting expression of ketohexokinase (KHK) in a cell, the agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having the formula (III): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-Y'Y'Y'-N ａ '-n ｑ '5' (IIIa) (In the formula: Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; the sense strand comprises at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; A double-stranded RNAi agent represented by:

74. A double stranded RNAi agent comprising the RNAi agent listed in any one of Tables 3, 4 5, 6, and 7.

75. 80. A cell comprising the double-stranded RNAi agent of any one of claims 1, 2, 7, 18, 67, or 69-74.

76. 1. A vector encoding at least one strand of a double-stranded RNAi agent, the double-stranded RNAi agent comprising a region of complementarity to at least a portion of an mRNA encoding a ketohexokinase, the double-stranded RNAi agent being 30 base pairs or less in length, and the double-stranded RNAi agent targets the mRNA for cleavage.

77. 77. The vector of claim 76, wherein the region of complementarity is at least 15 nucleotides in length.

78. 77. The vector of claim 76, wherein the region of complementarity is 19 to 21 nucleotides in length.

79. A cell comprising the vector of claim 76.

80. 77. A pharmaceutical composition comprising a double-stranded RNAi agent according to any one of claims 1, 2, 7, 18, 67 or 69-74, comprising a modified antisense polynucleotide agent according to claim 14 or a vector according to claim 76.

81. 81. The pharmaceutical composition of claim 80, wherein the double-stranded RNAi agent is administered in a non-buffered solution.

82. 82. The pharmaceutical composition of claim 81, wherein the non-buffered solution is saline or water.

83. 81. The pharmaceutical composition of claim 80, wherein the double-stranded RNAi agent is administered with a buffer.

84. 84. The pharmaceutical composition of claim 83, wherein the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

85. 85. The pharmaceutical composition of claim 84, wherein the buffer is phosphate buffered saline (PBS).

86. 1. A method for inhibiting ketohexokinase (KHK) expression in a cell, comprising: (a) contacting said cell with a double-stranded RNAi agent according to any one of claims 1, 2, 7, 18, 67 or 69-74, a pharmaceutical composition according to any one of claims 14-16 or 80-85, wherein said composition comprises a modified antisense polynucleotide agent according to claim 17 or a vector according to claim 76; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the KHK gene, thereby inhibiting expression of the KHK gene in the cells; A method comprising:

87. 87. The method of claim 86, wherein the cell is in a subject.

88. 88. The method of claim 87, wherein the subject is a human.

89. 89. The method of claim 88, wherein the subject is suffering from a disease associated with ketohexokinase.

90. The method of any one of claims 86 to 89, wherein the KHK expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

91. 13. A method of treating a subject suffering from a disorder associated with ketohexokinase (KHK), comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of any one of claims 1, 2, 7, 18, 67, 69-74, a pharmaceutical composition of any one of claims 14-16, or 80-85, thereby treating the subject, wherein the composition comprises a modified antisense polynucleotide agent of claim 17, or a vector of claim 76.

92. 1. A method of treating a subject suffering from a ketohexokinase (KHK) associated disorder, comprising subcutaneously administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, thereby treating the subject, the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides which differ from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides which differ from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by no more than 3 nucleotides; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end; substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end; wherein the sense strand is conjugated at its 3' end to one or more GalNAc derivatives attached via a branched divalent or trivalent linker.

93. 93. The method of claim 92, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

94. 93. The method of claim 91 or 92, wherein the subject is a human.

95. 95. The method of claim 94, wherein the ketohexokinase-associated disease is selected from the group consisting of liver disease, dyslipidemia, abnormal glycemic control, cardiovascular disease, kidney disease, metabolic syndrome, adipocyte dysfunction, visceral fat accumulation, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings.

96. 96. The method of claim 95, wherein the liver disease is fatty liver and / or steatohepatitis.

97. 96. The method of claim 95, wherein the dyslipidemia is selected from the group consisting of hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, and postprandial hypertriglyceridemia.

98. 96. The method of claim 95, wherein the abnormality in glycemic control is insulin resistance and / or diabetes.

99. 96. The method of claim 95, wherein the cardiovascular disease is hypertension and / or endothelial cell dysfunction.

100. 96. The method of claim 95, wherein the kidney disease is selected from the group consisting of acute kidney injury, tubular dysfunction, and inflammatory changes in the proximal tubules.

101. 93. The method of claim 91 or 92, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 1 mg / kg to about 10 mg / kg.

102. 102. The method of claim 101, wherein the double-stranded RNAi agent is administered at a dose of about 0.1 mg / kg, about 1.0 mg / kg, or about 3.0 mg / kg.

103. 102. The method of claim 101, wherein the double-stranded RNAi agent is administered at a dose of about 1 mg / kg to about 10 mg / kg.

104. 102. The method of claim 101, wherein the double-stranded RNAi agent is administered subcutaneously.

105. 102. The method of claim 101, wherein the double-stranded RNAi agent is administered intravenously.

106. 102. The method of claim 101, wherein the double-stranded RNAi agent is administered in two or more administrations.

107. 93. The method of claim 91 or 92, further comprising the step of administering to the subject an additional therapeutic agent.

108. 108. The method of claim 107, wherein the additional therapeutic agent is selected from the group consisting of an HMG-CoA reductase inhibitor, an antidiabetic agent, an antihypertensive agent, and resveratrol.

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