Ketohexokinase (KHK) iRNA composition and method of use thereof

Abstract

This invention provides compositions for treating diseases, disorders, and symptoms related to ketohexokinase (KHK) activity. [Solution] The present invention provides RNAi agents, such as dsRNA agents, that target the ketohexokinase (KHK) gene. The present invention also provides a method of using RNAi agents to inhibit KHK gene expression in a target, and a method for treating or preventing KHK-related diseases.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 62 / 732,600, filed on 18 September 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence List The present invention includes a sequence listing submitted in electronic format in ASCII format, which is incorporated herein by reference. A copy of the ASCII file was created on September 13, 2019, named 121301-06020_SL.txt, and is 309,325 bytes in size. [Background technology]

[0003] Epidemiological studies have shown that Western diets are one of the main causes of the modern obesity epidemic. Increased fructose intake is associated with the use of high-nutrient soft drinks and processed foods and is considered a major factor in the obesity epidemic. High-fructose corn syrup began to be widely used in the food industry by 1967. Although glucose and fructose have the same calorie value per molecule, these two sugars are metabolized via different metabolic pathways and utilize different GLUT transporters. Unlike the glucose metabolic pathway, fructose is almost exclusively metabolized in the liver, and the fructose metabolic pathway is not regulated by feedback inhibition by its products (Khaitan Z et al., (2013) J. Nutr. Metab. 2013, Article ID 682673, 1-12). Hexokinases and phosphofructokinases (PFKs) regulate the production of glyceraldehyde-3-P from glucose, while fructokinases or ketohexokinases (KHKs), which are responsible for the phosphorylation of fructose to fructose-1-phosphate in the liver, are not downregulated even when the concentration of fructose-1-phosphate increases. As a result, all fructose entering the cell is rapidly phosphorylated (Khaitan Z et al., (2013) J. Nutr. Metab. 2013, Article ID 682673, 1-12). The continuous use of ATP to phosphorylate fructose to fructose-1-phosphate leads to intracellular phosphate depletion, ATP depletion, activation of AMP deaminase, and uric acid formation (Khaitan Z. et al., (2013) J. Nutr. Metab. Article ID 682673, 1-12). Increased uric acid further promotes the upregulation of KHK (Lanaspa MA et al., (2012) PLOS ONE 7(10):1-11), leading to endothelial and adipocyte dysfunction. Subsequently, fructose-1-phosphate is converted to glyceraldehyde by aldolase B and then phosphorylated to glyceraldehyde-3-phosphate.The latter proceeds to the downstream glycolysis pathway, forming pyruvate and entering the citrate cycle. From there, under sufficient supply conditions, citrate is transported from the mitochondria to the cytosol, providing acetylcoenzyme A for lipid synthesis (Figure 1).

[0004] Phosphorylation of fructose by KHK, followed by activation of lipid synthesis, can lead to conditions such as fatty liver, hypertriglyceridemia, dyslipidemia, and insulin resistance. Inflammatory changes in renal proximal tubular cells have also been shown to be induced by KHK activity (Cirillo P. et al., (2009) J. Am. Soc. Nephrol. 20: 545-553). Phosphorylation of fructose by KHK is associated with liver diseases (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, type II diabetes), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction, etc.), kidney diseases (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, chronic kidney disease, etc.), metabolic syndrome, adipocyte dysfunction, visceral fat deposition, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings. Therefore, compositions and methods for treating diseases, disorders, and symptoms associated with KHK activity are needed in the art.

[0005] (Summary of the invention) The present invention provides compositions comprising RNAi agents that target ketohexokinase (KHK), such as double-stranded RNAi agents. The present invention also provides methods of using the compositions of the present invention to treat subjects with diseases that benefit from inhibiting KHK expression or reducing the expression of the KHK gene, such as liver diseases (e.g., fatty liver, steatohepatitis, especially non-alcoholic steatohepatitis (NASH)), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, type II diabetes, etc.), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction), kidney diseases (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, chronic kidney disease), metabolic syndrome, adipocyte dysfunction, visceral fat deposition, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings.

[0006] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of ketohexokinase (KHK), wherein the dsRNA comprises a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides, and the antisense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides.

[0007] In one embodiment, the sense strand is composed of nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 89 The sense strand comprises at least 15 consecutive nucleotides that differ from one of the following nucleotide sequences: 2-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, with three or fewer nucleotides differing from each other. In one embodiment, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1, or one of the aforementioned portions of the nucleotide sequence of SEQ ID NO: 1.

[0008] In one embodiment, in a particular embodiment, the sense strand and the antisense strand include nucleotide sequences selected from the group consisting of either one of the nucleotide sequences in Table 3 or Table 5.

[0009] In one embodiment, the sense chain or antisense chain is AD-72506, AD-72319, AD-72502, AD-72513, AD-72499, AD-72303, AD-72500, AD-72522, AD-72512, AD-72304, AD-72514, AD-72257, AD-72295, AD-72332, AD-72507, AD-72311, AD-72501, AD-725 It contains one double-stranded nucleotide sequence selected from the group consisting of 08, AD-72293, AD-72322, AD-72264, AD-72290, AD-72338, AD-72315, AD-72272, AD-72337, AD-72298, AD-72503, AD-72327, AD-72521, AD-72309, AD-72313, AD-72517, AD-72316, AD-72335, and AD-72317. In one embodiment, the sense chain and antisense chain are AD-72506, AD-72319, AD-72502, AD-72513, AD-72499, AD-72303, AD-72500, AD-72522, AD-72512, AD-72304, AD-72514, AD-72257, AD-72295, AD-72332, AD-72507, AD-72311, AD-72501, AD-725 It contains one double-stranded nucleotide sequence selected from the group consisting of 08, AD-72293, AD-72322, AD-72264, AD-72290, AD-72338, AD-72315, AD-72272, AD-72337, AD-72298, AD-72503, AD-72327, AD-72521, AD-72309, AD-72313, AD-72517, AD-72316, AD-72335, and AD-72317.

[0010] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of a ketohexokinase (KHK) gene, wherein the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in Table 3 or 5 by three or fewer nucleotides. In one embodiment, the dsRNA comprises a sense strand and an antisense strand, the antisense strand being AD-72506, AD-72319, AD-72502, AD-72513, AD-72499, AD-72303, AD-72500, AD-72522, AD-72512, AD-72304, AD-72514, AD-72257, AD-72295, AD-72332, AD-72507, AD-72311, AD-72501, AD-72508, AD-72293, AD-72322, AD-72264 It includes a complementary region containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one antisense sequence among any one of the double-stranded antisense sequences selected from the group consisting of AD-72290, AD-72338, AD-72315, AD-72272, AD-72337, AD-72298, AD-72503, AD-72327, AD-72521, AD-72309, AD-72313, AD-72517, AD-72316, AD-72335, or AD-72317.

[0011] In one embodiment, the dsRNA contains at least one modified nucleotide. In another embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand contain modifications.

[0012] In one embodiment, the present invention provides a double-stranded RNAi agent for inhibiting the expression of a ketohexokinase (KHK) gene, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprising at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 2, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end. In one embodiment, the sense strand is composed of nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-119 6, comprising at least 15 consecutive nucleotides differing by three or fewer nucleotides from one of the nucleotide sequences 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand comprises at least 15 consecutive nucleotides differing by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 2, where substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end. In one embodiment, the sense strand comprises at least 15 consecutive nucleotides of SEQ ID NO: 1, or any one of the aforementioned portions of the nucleotide sequence of SEQ ID NO: 1 and at least 15 consecutive nucleotides of the corresponding portion of SEQ ID NO: 2, wherein the sense strand and the antisense strand are complementary to each other.

[0013] In one embodiment, the present invention provides a double-stranded RNAi agent for inhibiting KHK expression, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, 959-977, 992-1010, 922-1 The sense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from one of the nucleotide sequences 041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 2, and the antisense strand is complementary to the sense strand's at least 15 consecutive nucleotides that differ by three or fewer nucleotides. In one embodiment, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end.

[0014] In one embodiment, the sense strand is composed of nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, 959-977, 992-1010, 922-1041, 1013-1041, and 1069 of SEQ ID NO: 1. The antisense strand comprises at least 15 consecutive nucleotides from one of the following sequences: ~1108, 1169~1140, 1111~1140, 1155~1196, 1221~1261, 1267~1294, or 1320~1350, wherein the antisense strand comprises at least 15 consecutive nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 2, and the antisense strand is complementary to at least 15 consecutive nucleotides of the sense strand, with three or fewer nucleotides being different. In one embodiment, substantially all of the nucleotides of the sense strand or substantially all of the nucleotides of the antisense strand are modified nucleotides, or substantially all of the nucleotides of both strands are modified nucleotides; the sense strand is conjugated to a ligand attached to its 3' end.

[0015] In one embodiment, the present invention provides a double-stranded RNAi agent for inhibiting KHK expression, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, The antisense strand comprises at least 15 consecutive nucleotides from one of the following sequences: 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand comprises at least 15 consecutive nucleotides from the corresponding position of the nucleotide sequence of SEQ ID NO: 2, and the antisense strand is complementary to at least 15 consecutive nucleotides of the sense strand. In one embodiment, substantially all nucleotides of the antisense strand are modified nucleotides. In one embodiment, substantially all nucleotides of both strands are modified. In a preferred embodiment, the sense strand is conjugated to a ligand attached to its 3' end.

[0016] In one embodiment, the antisense strand includes a complementary region comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences listed in either Table 3 or 5 by three or fewer nucleotides. For example, in one embodiment, the antisense strand may include AD-72506, AD-72319, AD-72502, AD-72513, AD-72499, AD-72303, AD-72500, AD-72522, AD-72512, AD-72304, AD-72514, AD-72257, AD-72295, AD-72332, AD-72507, AD-72311, AD-72501, AD-72508, AD-72293, AD-72322, AD-72264, AD-7 The antisense strand may include a complementary region containing at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one antisense sequence of any one of the double-stranded sequences selected from the group consisting of 2290, AD-72338, AD-72315, AD-72272, AD-72337, AD-72298, AD-72503, AD-72327, AD-72521, AD-72309, AD-72313, AD-72517, AD-72316, AD-72335, and AD-72317. In one embodiment, the antisense strand contains a region complementary to Sequence ID: 1, which contains at least 15 consecutive nucleotides from any one of the antisense sequences of the double-stranded sequence.

[0017] In some embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified.

[0018] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. In another embodiment, the modified nucleotide includes a short sequence of a 3'-terminal deoxythymine nucleotide (dT).

[0019] In one embodiment, substantially all nucleotides of the sense strand are modified. In another embodiment, substantially all nucleotides of the antisense strand are modified. In yet another embodiment, substantially all nucleotides of both the sense and antisense strands are modified.

[0020] In one embodiment, the double strand includes the modified antisense strand nucleotide sequence shown in Table 5. In another embodiment, the double strand includes the modified sense strand nucleotide sequence shown in Table 5. In yet another embodiment, the double strand includes the modified double strand shown in Table 5.

[0021] In one embodiment, the complementary region between the antisense strand and the target mRNA nucleotide sequence is at least 17 nucleotides long. For example, the complementary region between the antisense strand and the target is 19 to 21 nucleotides long, for example, the complementary region is 21 nucleotides long. In a preferred embodiment, each strand is 30 nucleotides or less in length.

[0022] In another embodiment, one or both strands of the double-stranded RNAi agent of the present invention have at least 19 consecutive nucleotide regions that are substantially complementary to at least a portion of the mRNA transcript of the KHK gene, and have a maximum nucleotide length of 66, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotide lengths. In some embodiments, the sense strand and the antisense strand form a double-stranded structure of 18-30 consecutive nucleotides.

[0023] In one embodiment, at least one strand of the dsRNA agent includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, at least one strand of the RNAi agent includes a 5' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 5' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of a single strand of the RNAi agent include an overhang of at least one nucleotide.

[0024] In one embodiment, the double-stranded RNAi agent further comprises a ligand. In one embodiment, the ligand is N-acetylgalactosamine (GalNAc). The ligand may be one or more GalNAcs conjugated to the RNAi agent via a monovalent, divalent, or trivalent branched linker. The ligand may be conjugated to the 3' end of the sense strand of the double-stranded RNAi agent, the 5' end of the sense strand of the double-stranded RNAi agent, the 3' end of the antisense strand of the double-stranded RNAi agent, or the 5' end of the antisense strand of the double-stranded RNAi agent.

[0025] In some embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of GalNAcs, for example, 2, 3, 4, 5, or 6 GalNAcs, each independently bound to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0026] In one embodiment, the ligand is [ka] That is the case.

[0027] In one embodiment, the double-stranded RNAi agent is conjugated to a ligand as shown in the schematic diagram below: [ka] (In the formula, X is either O or S). In one embodiment, X is O.

[0028] In one embodiment, the complementary region comprises one of the antisense nucleotide sequences from Table 3 or Table 5. In another embodiment, the complementary region comprises one of the antisense nucleotide sequences from Table 3 or Table 5.

[0029] In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of the KHK gene, the double-stranded RNAi agent comprising a sense strand, the sense strand comprising at least 15 consecutive nucleotides of any one of nucleotide sequences of nucleotides 89 to 107, 176 to 194, 264 to 282, 474 to 492, 508 to 526, 529 to 547, 562 to 580, 616 to 646, 682 to 700, 705 to 723, 705 to 757, 705 to 799, 739 to 757, 739 to 799, 760 to 799, 804 to 822, 837 to 855, 892 to 910, 959 to 977, 992 to 1010, 922 to 1041, 1013 to 1041, 1069 to 1108, 1169 to 1140, 1111 to 1140, 1155 to 1196, 1221 to 1261, 1267 to 1294 or 1320 to 1350 of SEQ ID NO: 1, the sense strand being complementary to the antisense strand, the antisense strand comprising a region complementary to a part of the mRNA encoding KHK, each strand being about 14 to about 30 nucleotides in length, and the dsRNA agent being of 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) (wherein, i, j, k and l are each independently 0 or 1; p, p', q and q' are each independently 0 to 6; each N a and N aEach of these sequences independently represents an oligonucleotide containing 0 to 25 nucleotides, which are either modified or unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b Each of these independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which can be modified, unmodified, or a combination thereof; each n p , n p ',n q and n q Each of these independently represents an overhang 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 Unlike the modification above Y, the modification above N b (The above modification differs 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 [formula].

[0030] 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. In another embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'. In another embodiment, the YYY motif is located at or near the cleavage site of the sense chain. In another embodiment, the YYY motif is located at positions 11, 12 and 13 from the 5' end of the antisense chain. In one embodiment, Y' is 2'-O-methyl.

[0031] For example, equation (III) is equation (IIIa): Sense: 5'np -N a -YYY-N a -n q 3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-n q '5' (IIIa) This can be shown by:

[0032] In another embodiment, equation (III) is equation (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 (' indicates an oligonucleotide sequence containing 1 to 5 modified nucleotides independently.) This can be shown by:

[0033] Alternatively, equation (III) is equation (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 (' indicates an oligonucleotide sequence containing 1 to 5 modified nucleotides independently.) This can be shown by:

[0034] Furthermore, equation (III) is given by equation (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 shows oligonucleotide sequences containing 1 to 5 modified nucleotides, each N a and N a ' independently indicates oligonucleotide sequences containing 2 to 10 modified nucleotides. This can be shown by:

[0035] In one embodiment, the double-stranded region has a length of 15 to 30 nucleotide pairs. For example, the double-stranded region may have a length of 17 to 23 nucleotide pairs. The double-stranded region may have a length of 17 to 25 nucleotide pairs. The double-stranded region may have a length of 23 to 27 nucleotide pairs. The double-stranded region may have a length of 19 to 21 nucleotide pairs. The double-stranded region may have a length of 21 to 23 nucleotide pairs.

[0036] In one embodiment, each chain has 15 to 30 nucleotides. In another embodiment, each chain has 19 to 30 nucleotides.

[0037] Modifications on the nucleotide can be selected from the group consisting of LNA, HNA, CeNA, 2-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In one embodiment, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0038] In one embodiment, the ligand is N-acetylgalactosamine (GalNAc). The ligand may be one or more GalNAcs conjugated to the RNAi agent via a monovalent, divalent, or trivalent branched linker. The ligand may be conjugated to the 3' end of the sense strand of the double-stranded RNAi agent, the 5' end of the sense strand of the double-stranded RNAi agent, the 3' end of the antisense strand of the double-stranded RNAi agent, or the 5' end of the antisense strand of the double-stranded RNAi agent.

[0039] In one embodiment, the double-stranded RNAi agent of the present invention comprises a plurality of GalNAcs, for example, 2, 3, 4, 5, or 6, each independently bound to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers. In one embodiment, the ligand is [ka] That is the case.

[0040] The ligand can bind to the 3' end of the sense strand.

[0041] An example of the structure of a dsRNAi agent conjugated to a ligand is shown in the following scheme: [ka] .

[0042] In one embodiment, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond. For example, the phosphorothioate or methylphosphonate internucleotide bond may be present at the 3' end of one of the strands, i.e., the sense strand or the antisense strand; or it may be present at the ends of both the sense strand and the antisense strand.

[0043] In one embodiment, the phosphorothioate or methylphosphonate internucleotide bond may be present at the 5' end of one strand, i.e., the sense strand or the antisense strand; or at both ends, i.e., the sense strand and the antisense strand.

[0044] In one embodiment, the phosphorothioate or methylphosphonate internucleotide bond may be present at both the 5' and 3' ends of one of the strands, i.e., the sense strand or the antisense strand; or it may be present at the ends of both strands, the sense strand and the antisense strand.

[0045] In one embodiment, the base pair at one position of the 5' end of the double-stranded antisense strand is an AU base pair.

[0046] In one embodiment, the Y nucleotide includes a 2'-fluoro modification. In another embodiment, the Y' nucleotide includes a 2'-O-methyl modification. In yet another embodiment, p'>0. In one embodiment, p'=2. In some embodiments, q'=0, p=0, q=0, and the p' overhang nucleotide is complementary to the target mRNA. In some embodiments, q'=0, p=0, q=0, and the p' overhang nucleotide is not complementary to the target mRNA.

[0047] In one embodiment, the sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides.

[0048] In one way, at least one n p' is attached to an adjacent nucleotide via a phosphorothioate bond. In other embodiments, all n p ' is attached to an adjacent nucleotide via a phosphorothioate bond.

[0049] In one embodiment, the dsRNAi agent is selected from the group of dsRNAi agents listed in Tables 3 and 5. In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified.

[0050] In one embodiment, the present invention provides a double-stranded RNAi agent capable of inhibiting KHK expression in cells, wherein the dsRNAi agent comprises a sense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, and 959 of SEQ ID NO: 1. The sense strand comprises at least 15 consecutive nucleotide segments of any one of the following: ~977, 992~1010, 922~1041, 1013~1041, 1069~1108, 1169~1140, 1111~1140, 1155~1196, 1221~1261, 1267~1294, or 1320~1350, wherein the sense strand is complementary to the antisense strand, the antisense strand comprises a region complementary to a portion of the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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 -Nb '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III)<0000�76>(wherein i, j, k and l are each independently 0 or 1; p, p', q and q' are each independently 0 to 6; each N a and N a ' each independently represent an oligonucleotide sequence containing 0 to 25 nucleotides that are either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' each independently represent an oligonucleotide sequence containing 0 to 10 nucleotides that are either modified or unmodified or a combination thereof; each n p , n p ', n q and n q ' each independently represent overhang nucleotides that may or may not be present; 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, and the modification is a 2-O-methyl modification or a 2'-fluoro modification; N b The modification on is different from the modification on Y, and the modification on N b ' is different from the modification on Y'; the sense strand is conjugated with at least one ligand) Provided is a double-stranded RNAi agent represented by.

[0051] In one embodiment, the present invention provides a double-stranded RNAi agent capable of inhibiting KHK expression in cells, wherein the dsRNA agent comprises a sense strand, which is preferably nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-85 5, comprising at least 15 consecutive nucleotide segments from one of the following: 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding KHK, and each strand is approximately 14-30 nucleotides long, where the dsRNA agent is given 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 either 0 or 1; each n p , n q and n q Each of these independently indicates an overhanging nucleotide, whether present or absent; p, q, and q' are each independently between 0 and 6; n p '>0 and at least one n p ' is attached to an adjacent nucleotide via a phosphorothioate bond; each N a and N a Each of these independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides that are either modified, unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one of three identical modification motifs on three consecutive nucleotides, the modification being either a 2'-O-methyl modification or a 2'-fluoro modification; N b Unlike the modification above Y, the modification above N b The above modifier differs from the above modifier; The sense strand is conjugated with at least one ligand. The present invention provides a double-stranded RNAi agent represented by [formula].

[0052] In one embodiment, the present invention provides a double-stranded RNAi agent capable of inhibiting KHK expression in cells, wherein the double-stranded RNAi agent comprises a sense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, 959-9 The sense strand comprises at least 15 consecutive nucleotide segments of any one of the following nucleotides: 77, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the sense strand is complementary to the antisense strand, and the antisense strand contains a region complementary to a portion of the mRNA encoding KHK, with each strand having a length of approximately 14-30 nucleotides, where the dsRNA agent is given 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 either 0 or 1; each n p , n q and n qEach of these independently indicates an overhanging nucleotide, whether present or absent; p, q, and q' are each independently between 0 and 6; n p '>0 and at least one n p ' is attached to an adjacent nucleotide via a phosphorothioate bond; each N a and N a Each of these independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which are either modified, unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one of three identical modification motifs on three consecutive nucleotides, where the modification is either a 2'-O-methyl modification or a 2'-fluoro modification; N b Unlike the modification above Y, the modification above N b The above modifier differs from the above modifier; The sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker. The present invention provides a double-stranded RNAi agent as shown in [the provided text].

[0053] In one embodiment, the present invention provides a double-stranded RNAi agent capable of inhibiting KHK expression in cells, wherein the dsRNA agent comprises a sense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, and 959 of SEQ ID NO: 1. The sense strand comprises at least 15 consecutive nucleotide segments of any one of the following: ~977, 992~1010, 922~1041, 1013~1041, 1069~1108, 1169~1140, 1111~1140, 1155~1196, 1221~1261, 1267~1294, or 1320~1350, wherein the sense strand is complementary to the antisense strand, which contains a region complementary to a portion of the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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 either 0 or 1; each n p , n q and n q Each of these independently indicates an overhanging nucleotide, whether present or absent; p, q, and q' are each independently between 0 and 6; n p '>0 and at least one n p ' is attached to an adjacent nucleotide via a phosphorothioate bond; each N a and N a Each of these independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b Each of these independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which are either modified, unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one of three identical modification motifs on three consecutive nucleotides, where the modification is either a 2'-O-methyl modification or a 2'-fluoro modification; N b Unlike the modification above Y, the modification above N b The above modifier differs from the above modifier; The sense chain contains at least one phosphorothioate bond; The sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker. The present invention provides a double-stranded RNAi agent as shown in [the provided text].

[0054] In one embodiment, the present invention provides a double-stranded RNAi agent capable of inhibiting KHK expression in cells, wherein the dsRNA agent comprises a sense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, and 959 of SEQ ID NO: 1. The sense strand comprises at least 15 consecutive nucleotide segments of any one of the following: ~977, 992~1010, 922~1041, 1013~1041, 1069~1108, 1169~1140, 1111~1140, 1155~1196, 1221~1261, 1267~1294, or 1320~1350, wherein the sense strand is complementary to the antisense strand, and the antisense strand contains a region complementary to a portion of the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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 p , n q and n q Each of these independently indicates an overhanging nucleotide, whether present or absent; p, q, and q' are each independently between 0 and 6; n p '>0 and at least one n p ' is attached to an adjacent nucleotide via a phosphorothioate bond; each N a and N aEach of these independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one of three identical modification motifs on three consecutive nucleotides, the modification being either a 2'-O-methyl modification or a 2'-fluoro modification; The sense chain contains at least one phosphorothioate bond; The sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker. The present invention provides a double-stranded RNAi agent as shown in [the provided text].

[0055] In one embodiment, the present invention provides a double-stranded RNAi agent for inhibiting KHK expression, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides, for example, nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-5 of SEQ ID NO: 1 47, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294 or 1320-1350 The sense strand contains 15 consecutive nucleotides that differ from any one nucleotide sequence by three or fewer nucleotides, the antisense strand contains at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides, substantially all nucleotides of the sense strand contain modifications selected from 2'-O-methyl and 2'-fluoro modifications, the sense strand contains two phosphorothioate nucleotide interlinks at its 5' end, substantially all nucleotides of the antisense strand contain modifications selected from 2'-O-methyl and 2'-fluoro modifications, the antisense strand contains two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end, and the sense strand is conjugated to one or more GalNAc derivatives linked via monovalent, or divalent or trivalent linkers of branched branches.In one embodiment, the sense strand comprises at least 15 consecutive nucleotides of SEQ ID NO: 1 or one of the aforementioned portions of SEQ ID NO: 1 and at least 15 consecutive nucleotide sequences of the corresponding portion of SEQ ID NO: 2, wherein the antisense strand is complementary to at least 15 consecutive nucleotides, with three or fewer nucleotides in the sense strand being different. In one embodiment, the sense strand and the antisense strand comprise at least 15 consecutive nucleotides of SEQ ID NO: 1 and SEQ ID NO: 2, or comprise one of a predetermined portion of SEQ ID NO: 1 and the corresponding portion of SEQ ID NO: 2.

[0056] In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In another embodiment, each strand has 19 to 30 nucleotides.

[0057] In one embodiment, substantially all nucleotides of the sense strand are modified. In another embodiment, substantially all nucleotides of the antisense strand are modified. In yet another embodiment, substantially all nucleotides of both the sense and antisense strands are modified.

[0058] In one embodiment, the present invention provides cells comprising the dsRNA agent described herein.

[0059] In one embodiment, the present invention provides a vector encoding at least one strand of a dsRNA agent, wherein the antisense strand includes a region complementary to at least a portion of the mRNA encoding KHK, the dsRNA being 30 base pairs or less in length, and the dsRNA agent targets the mRNA for cleavage. In one embodiment, the complementary region is at least 15 nucleotides long. In another embodiment, the complementary region is 19 to 23 nucleotides long.

[0060] In one embodiment, the present invention provides cells comprising the vector described herein.

[0061] In one embodiment, the present invention provides a pharmaceutical composition for inhibiting the expression of the KHK gene, comprising the dsRNA agent of the present invention. In one embodiment, the dsRNA agent is administered in a non-buffered solution. In one embodiment, the non-buffered solution is physiological saline or water. In another embodiment, the dsRNA agent is administered with a buffer. In such an embodiment, the buffer may contain acetate, citrate, prolamin, carbonate, phosphate, or any combination thereof. For example, the buffer may be phosphate-buffered saline (PBS).

[0062] In one embodiment, the present invention provides a pharmaceutical composition comprising the dsRNA agent and a lipid preparation. In one embodiment, the lipid preparation comprises LNP. In another embodiment, the lipid preparation comprises MC3.

[0063] In one embodiment, the present invention provides a method for inhibiting KHK expression in cells, the method comprising: (a) contacting cells with the dsRNA agent of the present invention or the pharmaceutical composition of the present invention; and (b) maintaining the cells that have undergone step (a) for a sufficient time to obtain degradation products of the mRNA transcript of the KHK gene, thereby inhibiting the expression of the KHK gene in the cells. In one embodiment, the cells are present in a subject, e.g., a human subject, e.g., a female or a male. In one embodiment, the subject has impaired renal function or shows a tendency toward impaired renal function. In a preferred embodiment, KHK expression is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to a suitable control, or reduced to below the detection limit. In one embodiment, the sense strand contains at least 15 consecutive nucleotides having three or fewer mismatches with SEQ ID NO: 1, or nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892- The antisense strand contains at least 15 consecutive nucleotides from one of the following sequences: 910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350 and the corresponding portion of SEQ ID NO: 2, wherein the antisense strand is complementary to at least 15 consecutive nucleotides in the sense strand, with three or fewer nucleotides being different. In one embodiment, the sense strand and the antisense strand contain at least 15 consecutive nucleotide sequences from SEQ ID NO: 1 and SEQ ID NO: 2, or contain one of the predetermined portions of SEQ ID NO: 1 and the corresponding portion of SEQ ID NO: 2, wherein the antisense strand is complementary to at least 15 consecutive nucleotide sequences in the sense strand, with three or fewer nucleotides being different.

[0064] In one embodiment, the present invention provides a method for treating a subject suffering from a disease or disorder that benefits from reduced KHK expression, the method characterized by treating the subject by administering a therapeutically effective amount of the dsRNA agent or pharmaceutical composition of the present invention to the subject.

[0065] In one embodiment, the present invention provides a method for preventing at least one symptom in a subject suffering from a disease or disorder that benefits from reduced KHK expression, the method being characterized by preventing at least one symptom in a subject suffering from a disorder that benefits from reduced KHK expression by administering a prophylactically effective amount of the dsRNA agent or pharmaceutical composition of the present invention to the subject.

[0066] In one embodiment, administration of a dsRNA agent to a patient causes a decrease in fructose metabolism. In one embodiment, administration of dsRNA causes a decrease in KHK levels, particularly hepatic KHK, and especially KHK-C, in subjects exhibiting high KHK levels. In one embodiment, administration of dsRNA causes a decrease in fructose metabolism in subjects. In one embodiment, administration of dsRNA causes a decrease in uric acid levels (e.g., serum uric acid) in subjects exhibiting high serum uric acid levels (e.g., subjects exhibiting high serum uric acid levels associated with gout). In one embodiment, administration of dsRNA causes normalization of serum lipids, e.g., triglycerides including postprandial triglycerides, LDL, HDL, or cholesterol, in subjects having at least one abnormal serum lipid level. In one embodiment, administration of dsRNA causes normalization of lipid deposition, e.g., a decrease in hepatic lipid deposition (e.g., a decrease in NAFLD or NASH), a decrease in visceral fat deposition, and a decrease in body weight. In one embodiment, administration of dsRNA normalizes the insulin response or glucose response in subjects exhibiting an abnormal insulin response or glucose response unrelated to an immune response to insulin. In another embodiment, administration of dsRNA improves renal function or prevents or reduces the rate of renal function loss. In yet another embodiment, dsRNA reduces hypertension, i.e., elevated blood pressure.

[0067] In one embodiment, KHK-related disease is liver disease, such as fatty liver disease like NAFLD or NASH. In another embodiment, KHK-related disease is dyslipidemia, such as elevated serum triglycerides, elevated serum LDL, elevated serum cholesterol, decreased serum HDL, or postprandial hypertriglyceridemia. In yet another embodiment, KHK-related disease is impaired glycemic control, such as insulin resistance not caused by an immune response to insulin, glucose resistance, or type II diabetes. In one embodiment, KHK-related disease is cardiovascular disease, such as hypertension or endothelial cell dysfunction. In one embodiment, KHK-related disease is kidney disease, such as acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, or chronic kidney disease. In one embodiment, the disease is metabolic syndrome. In one embodiment, KHK-related disease is lipid deposition or dysfunction, such as visceral fat deposition, fatty liver, or obesity. In one embodiment, KHK-related disorders are diseases related to elevated uric acid levels, such as gout and hyperuricemia. In another embodiment, KHK-related disorders are eating disorders such as excessive sugar cravings.

[0068] In one embodiment, the present invention further includes administering an additional agent to a subject having a KHK-related disease.

[0069] In one embodiment, treatments known to those skilled in the art for various KHK-related diseases are used in combination with the RNAi agents of the present invention. Such treatments are discussed below.

[0070] In various embodiments, the dsRNAi agent is administered to the subject in doses of approximately 0.01 mg / kg to approximately 10 mg / kg, or approximately 0.5 mg / kg to approximately 50 mg / kg. In some embodiments, the dsRNAi agent is administered to the subject in doses of approximately 10 mg / kg to approximately 30 mg / kg. In one embodiment, the dsRNAi agent is administered to the subject in doses selected from 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg. In one embodiment, the RNAi agent is administered in doses of approximately 0.1 mg / kg to approximately 5.0 mg / kg, once approximately once a week, once approximately once a month, once approximately every two months, or once approximately every quarter (i.e., once approximately every three months).

[0071] In one embodiment, the dsRNAi agent is administered to the subject once a week. In another embodiment, the dsRNAi agent is administered to the subject once a month. In yet another embodiment, the dsRNAi agent is administered to the subject once a quarter (i.e., once every three months).

[0072] In one embodiment, the dsRNAi agent is administered subcutaneously to the subject.

[0073] In one embodiment, the dsRNAi agent is administered intramuscularly to the subject.

[0074] In various embodiments, the method of the present invention further includes measuring the uric acid level, particularly the serum uric acid level, of the subject. In various embodiments, the method of the present invention further includes measuring the uric acid fluctotose level in the subject. In various embodiments, the method of the present invention further includes measuring the serum lipid level in the subject. In one embodiment, the method of the present invention further includes measuring insulin or glucose sensitivity in the subject. In one embodiment, a decrease in the expression or activity level of fructose metabolism indicates that KHK-related disease is being treated or prevented. [Brief explanation of the drawing]

[0075] [Figure 1] Figure 1 shows the classical and alternative lipid synthesis pathways for fructose. In the classical pathway, triglycerides (TG) are the direct products of fructose metabolism through the action of multiple enzymes, including aldolase B (Aldo B) and fatty acid synthase (FAS). In the alternative pathway, uric acid, produced by nucleotide turnover during the phosphorylation of fructose to fructose-1-phosphate (F-1-P), creates mitochondrial oxidative stress (mtROS), which reduces the activity of aconitase (ACO2) in the Krebs cycle. As a result, citrate, a substrate of ACO2, accumulates and is released into the cytosol, where it acts as a substrate for TG synthesis through the activation of ATP citrate lyase (ACL) and fatty acid synthase. AMPD2, AMP deaminase 2; IMP, inosine monophosphate; PO4, phosphate (Jonshon et. al., (2013) Diabetes. 62:3307-3315). [Figure 2] Figure 2 shows the exon configurations in the human KHK gene for the transcripts of ketohexokinase A (NM_000221.2, SEQ ID NO: 3), ketohexokinase C (NM_006488.2, SEQ ID NO: 1), and transcription mutant X5 (XM_005264298.1, SEQ ID NO: 5). [Modes for carrying out the invention]

[0076] The present invention provides a composition comprising an RNAi agent that targets KHK, such as a double-stranded iRNA agent. The present invention also provides a method of using the composition of the present invention to inhibit KHK expression, as well as a method of using the composition of the present invention to treat KHK-related diseases, disorders and / or symptoms, such as liver diseases (e.g., fatty liver, steatohepatitis, NAFLD, NASH), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance not due to immune response to insulin, type II diabetes), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction), kidney diseases (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, chronic kidney disease), metabolic syndrome, adipocyte dysfunction, visceral fat deposition, obesity, hyperuricemia, gout, eating disorders and excessive sugar cravings (Khaitan Z. et al., (2013) J. Nutr. Metab., Article ID 682673). 1-12;Diggle CP et al., (2009) J. Hisotchem. Cytochem., 57(8): 763-774;Cirillo P. et al., (2009) J. Am. Soc. Nephrol., 20: 545-553;Lanaspa MA et al., (2012) PLOS ONE 7(10): 1-11).

[0077] The KHK (ketohexokinase) gene is located on chromosome 2p23 and encodes ketohexokinase, also known as fructokinase. KHK is a phosphotransferase enzyme that uses alcohol as its 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 obtained by alternative splicing of full-length mRNA. These isoforms differ in whether they contain exon 3a or 3c, and differ in 32 amino acids at positions 72-115 (see, for example, Figure 2). KHK-C mRNA is expressed at high levels mainly in the liver, kidney, and small intestine. KHK-C has a much lower K2 receptor for fructose binding than KHK-A. m It possesses and, as a result, is highly effective in phosphorylating dietary fructose. The sequence of the human KHK-C mRNA transcript can be found, for example, at GenBank registry number GI:153218447 (NM_006488.2; SEQ ID NO: 1). The sequence of the human KHK-A mRNA transcript can be found, for example, at GenBank registry number GI:153218446 (NM_000221.2; SEQ ID NO: 3). The sequence of the full-length human KHK mRNA is provided at GenBank registry number GI:530367552 (XM_005264298.1; SEQ ID NO: 5), and this was used (Figure 2).

[0078] This invention provides iRNA agents, compositions, and methods for regulating the expression of the KHK gene. In one embodiment, KHK expression is reduced using a KHK-specific iRNA agent, thereby reducing the phosphorylation of fructose to fructose-1-phosphate, preventing an increase in uric acid levels and lipid synthesis, and leading to metabolism via the fructose metabolic pathway. Thus, inhibition of KHK gene expression or activity using the iRNA composition of this invention is useful as a therapeutic method to reduce the lipid synthesis effect of dietary fructose and prevent the resulting accumulation of uric acid in the subject. Such inhibition is useful in treating diseases, disorders, or symptoms such as liver diseases (e.g., fatty liver, steatohepatitis, NAFLD, NASH), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes mellitus), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction), kidney diseases (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, chronic kidney disease), metabolic syndrome, adipocyte dysfunction, visceral fat deposition, obesity, hyperuricemia, gout, eating disorders, and excessive sugar cravings.

[0079] This invention provides iRNA compositions that influence RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the ketohexokinase (KHK) gene. This gene may be present in cells within a target organism, such as human cells. By using these iRNAs, targeted degradation of the mRNA of the corresponding gene (KHK gene) becomes possible in mammals.

[0080] The iRNAs of the present invention are designed to target human KHK genes, including portions of the gene that are conserved in KHK orthologues of other mammals. While not intended to be theoretically limited, it is believed that the aforementioned properties, along with specific target sites or specific modifications in these iRNAs, or any combination or partial combination thereof, contribute to the improved efficacy, stability, potency, durability, and safety of the iRNAs of the present invention.

[0081] Accordingly, the present invention also provides a method for treating subjects with disorders that benefit from inhibiting or reducing the expression of the KHK gene, such as KHK-related diseases, using an iRNA composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the KHK gene.

[0082] At very low doses, the iRNA of the present invention can specifically and efficiently mediate RNA interference (RNAi), and as a result, significantly inhibit the expression of the corresponding gene (KHK gene).

[0083] The iRNA of the present invention is an RNA chain (antisense chain) having a region with a nucleotide length of approximately 30 or less, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 1 The region may contain nucleotide lengths of 9-27, 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, and this region is substantially complementary to at least a portion of the mRNA transcript of the KHK gene.

[0084] In one embodiment, the iRNA of the present invention includes an RNA strand (antisense strand) that may have a longer nucleotide length, for example, up to 66 nucleotides, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides, and may contain a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the KHK gene. These iRNAs having a longer antisense strand length preferably include a second RNA strand (sense strand) of 20-60 nucleotides in length, where the sense strand and the antisense strand form a double helix of 18-30 consecutive nucleotides.

[0085] The use of the iRNA of the present invention enables targeted degradation of the mRNA of the corresponding gene (KHK gene) in mammals. At very low doses, the iRNA of the present invention can specifically and efficiently mediate RNA interference (RNAi), thereby significantly inhibiting the expression of the corresponding gene (KHK gene). Using in vitro and in vivo assays, we demonstrated that iRNAs targeting the KHK gene can mediate RNAi, resulting in significant inhibition of KHK expression and reduced fructose metabolism, thereby alleviating one or more symptoms associated with KHK-related diseases. Therefore, methods and compositions containing these iRNAs are useful for treating subjects suffering from KHK-related diseases. The methods and compositions described herein are useful for reducing the level of KHK, preferably KHK-C, in the subject, for example, the level of KHK-C in the liver of the subject.

[0086] The following detailed description discloses methods for producing and using compositions comprising iRNA for inhibiting KHK gene expression, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from reduced KHK gene expression.

[0087] I. Definition To facilitate understanding of this invention, several terms are first defined. Furthermore, it should be noted that whenever parameter values ​​or ranges of values ​​are described, intermediate values ​​and ranges of those values ​​are also intended to be part of this invention.

[0088] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements, e.g., multiple elements.

[0089] The term "including" is used herein to mean "including, but not limited to," and is used interchangeably with this term.

[0090] The term "or" is used herein to mean and is interchangeable with the term "and / or" unless the context clearly indicates otherwise.

[0091] The term “approximately” is used herein to mean a typical range of acceptable ranges in the art. For example, “approximately” may be understood as a standard deviation of about 2 from the mean. In one embodiment, “approximately” means ±10%. In another embodiment, “approximately” means ±5%. When “approximately” precedes a set of numbers or ranges, it is understood that “approximately” can modify each number within that set of numbers or ranges.

[0092] The term "at least" preceding a number or a range of numbers is understood, as is clear from the context, to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a nucleic acid molecule with 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the given property. When "at least" is present before a range of numbers or numbers, it is understood that "at least" can modify each number in the range of numbers or numbers.

[0093] As used herein, “less than” or “less than” is understood from the context as a logical value down to zero, as a number and logically lower value or integer adjacent to this term. For example, a double helix with an overhang of “2 or fewer nucleotides” has an overhang of 2, 1, or 0 nucleotides. If “less than” precedes a number or range, it is understood that “less than” can modify each number in the number or range.

[0094] As used herein, “ketohexokinase” or “KHK” is an enzyme that catalyzes the conversion of fructose to fructose-1-phosphate. The gene product is the first enzyme in the pathway that catabolizes dietary fructose. Selective transcriptional variants encoding various isoforms have been identified. This gene is also known as fructokinase. Further information regarding KHK is available, for example, in the NCBI gene database (www.ncbi.nlm.nih.gov / gene / 3975) (incorporated herein by reference as of the filing date).

[0095] As used herein, the term "ketohexokinase," used interchangeably with the term "KHK," refers to the naturally occurring gene that codes for the KHK protein. The amino acid and complete coding sequences of the human KHK gene reference sequence may be found, for example, in GenBank registry numbers GI:153218447 (RefSeq registry number NM_006488; SEQ ID NO: 1; SEQ ID NO: 2), GenBank registry number GI:153218446 (RefSeq registry number NM_000221.2; SEQ ID NOs: 3 and 4), and GenBank registry number 767914480 (RefSeq registry number XM_005264298.2; SEQ ID NOs: 5 and 6). Mammalian autologs of the human KHK gene can be found, for example, in GI:887209819 (RefSeq registry number NM_008439.4, mouse; SEQ ID NO: 7 and SEQ ID NO: 8); GI:126432547 (RefSeq registry number NM_031855.3, rat; SEQ ID NO: 9 and SEQ ID NO: 10); and GenBank registry number GI:982291245 (RefSeq registry number XM_005576321, cynomolgus monkey; SEQ ID NO: 11 and SEQ ID NO: 12).

[0096] Two KHK isoforms exist, produced by alternative splicing of KHK premRNA. KHK-C is abundant in organs involved in fructose metabolism, such as the liver, kidneys, and intestines. It is highly active and involved in most fructose metabolism. KHK-A has a low affinity for fructose and is widely expressed in most tissues. The iRNA agents provided herein can silence one or both KHK isoforms. In a preferred embodiment, the iRNA agent is capable of silencing at least KHK-C, thereby inhibiting the expression of at least the KHK-C isoform.

[0097] Many naturally occurring SNPs are known and can be found, for example, in the NCBI SNP database (www.ncbi.nlm.nih.gov / SNP / snp_ref.cgi?locusId=3795), which provides SNPs in human KHK (as of the filing date, incorporated herein by reference). In a preferred embodiment, such naturally occurring variants fall within the range of the KHK gene sequence.

[0098] Examples of KHK mRNA sequences can be readily obtained using well-known databases, such as GenBank, UniProt, and OMIM.

[0099] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a KHK gene, such as the mRNA that is the product of RNA processing of the primary transcript. The target portion of the sequence will be long enough to serve as a substrate for iRNA-specific cleavage, at least in or near the relevant portion of the nucleotide sequence of the mRNA molecule formed during the transcription of a KHK gene. In one embodiment, the target sequence is located within the protein-coding region of KHK.

[0100] 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 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23 or 21 to 22 nucleotides in length. The lengths of the above ranges and the ranges therebetween are also considered to be part of the present invention.

[0101] As used herein, the term "strand containing a sequence" refers to an oligonucleotide containing a nucleotide strand represented by a sequence shown using standard nucleotide nomenclature.

[0102] "G", "C", "A", and "U" generally each represent a nucleotide containing guanine, cytosine, adenine, and uracil as bases, respectively. However, the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or surrogate replacement moieties, as will be described in more detail below (see, for example, Table 2). Those skilled in the art will fully recognize that guanine, cytosine, adenine, and uracil may be replaced by another replacement moiety that does not substantially change the base pairing properties of the oligonucleotide containing nucleotides having such replacement moieties. For example, without limitation, 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 replaced, for example, with a nucleotide containing inosine in the nucleotide sequence of the dsRNA according to the invention. In another example, adenine and cytosine at any position in an oligonucleotide can be replaced with guanine and uracil, respectively, to form wobble G-U base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods described in the present invention.

[0103] The terms "iRNA", "RNAi agent", "iRNA agent", and "RNA interference agent", which are used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of an RNA transcript via a pathway through an RNA-induced silencing complex (RISC), as the terms are defined herein. iRNA leads to sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of the KHK gene in cells, such as cells of a mammalian subject.

[0104] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a KHK target mRNA sequence, to lead to the cleavage of the target RNA. Although we do not wish to be limited to theory, it is thought that long double-stranded RNA introduced into a cell is degraded into siRNA 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 short interfering RNAs of 19-23 base pairs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to be directed towards target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the present invention relates to single-stranded RNA (siRNA) that is generated in cells and facilitates the formation of a RISC complex that silencing a target gene, namely the KHK gene. Accordingly, the term "siRNA" is also used herein to refer to such iRNA.

[0105] In one embodiment, the RNAi agent may be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute2 and then cleaves the target mRNA. Single-stranded siRNAs typically consist of 15 to 30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, all of which are incorporated herein by reference. Any antisense nucleotide sequence described herein may be used as a single-stranded siRNA as described herein or as a single-stranded siRNA chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0106] In one embodiment of the present invention, the “iRNA” used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are shown to have “sense” and “antisense” orientations toward the target RNA (i.e., the KHK gene). In one embodiment 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 herein to as RNA interference or RNAi.

[0107] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, as used herein, “iRNA agents” may contain ribonucleotides having chemical modifications; iRNA agents may contain substantial modifications of multiple nucleotides. As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified internucleotide bond, or a modified nucleic acid base or a combination thereof. Thus, the term modified nucleotide includes, for example, substitution, addition, or removal of functional groups or atoms to internucleoside bonds, sugar moieties, or nucleic acid bases. 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 modification when used in siRNA-type molecules is encompassed in “iRNA” or “RNAi agent” for the purposes of this specification and the claims.

[0108] While the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides, as described in detail herein. Furthermore, as used herein, “iRNA” may include ribonucleotides having chemical modifications; an iRNA agent may include substantial modifications of multiple nucleotides. As used herein, the term “modified nucleotide” independently refers to a nucleotide having a modified sugar moiety, a modified internucleotide bond, or a modified nucleic acid base. Thus, the term modified nucleotide encompasses, for example, the substitution, addition, or removal of functional groups or atoms to internucleoside bonds, sugar moieties, or nucleic acid bases. Suitable modifications for use in the agents of the present invention include any type of modification disclosed herein or known in the art. Any such modification when used in siRNA-type molecules is encompassed in “iRNA” or “RNAi agent” for the purposes of this specification and the claims.

[0109] The double-stranded region may be of any length that enables the specific degradation of the desired target RNA via the RISC pathway, such as approximately 9 to 36 base pairs in length, for example, approximately 15 to 30 base pairs in length, for example, approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 base pairs in length, for example, approximately 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19- The length may be in the range of 20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths beyond those listed above are also considered part of the present invention.

[0110] The two strands forming a 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 joined by a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, this joined RNA strand is called a “hairpin loop.” A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 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 twenty, at least 23 or more unpaired nucleotides. In some embodiments, a hairpin loop may contain ten or more unpaired nucleotides. In some embodiments, a hairpin loop may contain eight or more unpaired nucleotides. In some embodiments, a hairpin loop may contain four to ten unpaired nucleotides. In some embodiments, a hairpin loop may contain four to eight unpaired nucleotides.

[0111] When two substantially complementary strands of dsRNA are composed of other RNA molecules, these molecules may, but do not necessarily, be covalently linked. If the two strands are covalently linked by means other than a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, this linking structure is called a "linker." 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 dsRNA minus any overhangs present in the double helix. In addition to the double helix structure, RNAi may contain one or more nucleotide overhangs.

[0112] In one embodiment, the iRNA agent of the present invention is a dsRNA, each strand containing 19-23 nucleotides, which interacts with a target RNA sequence (e.g., the KHK gene). Although we do not wish to be bound by theory, the long double-stranded RNA introduced into the cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a type III ribonuclease enzyme, processes the dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Subsequently, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, enabling the complementary antisense strand to be guided towards target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0113] In some embodiments, the iRNA of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a KHK target mRNA sequence, to cleave the target RNA. While we do not wish to be bound by theory, long double-stranded RNA introduced into a cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a type III ribonuclease enzyme, processes the dsRNA into a short interfering RNA of 19-23 base pairs with a characteristic 3' overhang of two bases (Bernstein, et al., (2001) Nature 409:363). Subsequently, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to be guided towards target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0114] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide protruding from a double-stranded iRNA. For example, a nucleotide overhang exists if the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or 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 contain or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located at the 5' end, the 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0115] In one embodiment, the antisense strand of dsRNA has an overhang of 1 to 10 nucleotides at its 3' and / or 5' ends, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. The overhang on the sense strand or the antisense strand, or both, may encompass a length of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides. In one embodiment, the extended overhang may encompass a self-complementary portion, i.e., the overhang may encompass a double helix of at least 3 nucleotides or at least 4 nucleotides, which can form a stable hairpin structure. In one embodiment, the extended overhang is located on a double-stranded sense strand. In one embodiment, the extended overhang is located on the 3' end of a double-stranded sense strand. In one embodiment, the extended overhang is located on the 5' end of a double-stranded sense strand. In one embodiment, the extended overhang is located on the double-stranded antisense strand. In another embodiment, the extended overhang is located on the 3' end of the double-stranded antisense strand. In yet another embodiment, the extended overhang is located on the 5' end of the double-stranded antisense strand. In yet another embodiment, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates. In yet another embodiment, the overhang comprises a self-complementary portion that can form a stable hairpin structure under physiological conditions.

[0116] "Bluish" or "blunt-ended" means that there are no unpaired nucleotides at the corresponding end of a double-stranded RNAi agent, i.e., there are no nucleotide overhangs. A "blunt-ended" double-stranded RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents that have no nucleotide overhang at one end (i.e., agents having one overhang and one blunt end) or RNAi agents that have no nucleotide overhangs at both ends.

[0117] The terms “antisense strand” or “guide strand” refer to, for example, a strand of iRNA, e.g., dsRNA, that contains a region substantially complementary to the target sequence, e.g., KHK mRNA. As used herein, the term “complementary region” refers to a region of the antisense strand that is substantially complementary to the sequence, e.g., the target sequence, e.g., KHK nucleotide sequence, as defined herein. If the complementary region is not fully complementary to the target sequence, mismatches may exist in the internal or terminal regions of the molecule. Generally, most acceptable mismatches are located in terminal regions, e.g., in 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the iRNA. In some embodiments, the double-stranded RNAi agent of the present invention encompasses nucleotide mismatches in the antisense strand. In some embodiments, the double-stranded RNAi agent of the present invention encompasses nucleotide mismatches in the sense strand. In some embodiments, the nucleotide mismatch is located, for example, in 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is located, for example, at the 3' end of the iRNA.

[0118] As used herein, the terms “sense strand” or “passenger strand” refer to a strand of iRNA that contains a region substantially complementary to the antisense strand region, as defined herein.

[0119] As used herein, “substantially all nucleotides are modified” means that a wide range of nucleotides are modified, but not all, and may include 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0120] As used herein, the term “cleavage region” refers to a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where cleavage occurs. In one embodiment, the cleavage region includes three bases directly adjacent to the cleavage site at either end of the cleavage site. In another embodiment, the cleavage region includes two bases directly adjacent to the cleavage site at either end of the cleavage site. In yet another embodiment, the cleavage site occurs specifically at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0121] As used herein, unless otherwise stated, the term “complementary” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under given conditions to form a double-stranded structure, when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be stringent conditions, for example, including 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 50°C or 70°C for 12–16 hours, and subsequent 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 within living organisms, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of the two sequences, depending on the final use of the hybridized nucleotides.

[0122] In the iRNAs described herein, for example, complementary sequences in dsRNAs include base pairings of one or both nucleotide sequences over the full length of one or both nucleotide sequences of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences can be said herein to be “fully complementary” to each other. However, where the first sequence is said herein to be “substantially complementary” to the second sequence, the two sequences may be fully complementary, or, when hybridized to double helixes of up to 30 base pairs, while retaining the ability to hybridize under conditions optimal for their end use, for example, under conditions optimal for inhibiting gene expression via the RISC pathway, they may form one or more mismatched base pairs, but generally five or fewer, four or fewer, three or fewer, or two or fewer. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and the other oligonucleotide of 23 nucleotides for the purposes described herein can be said to be "perfectly complementary" if the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide.

[0123] As used herein, “complementary” sequences may include, or may be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, provided that the above requirements relating to their hybridizing ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation type or Hoogsteen type base pairs.

[0124] As used herein, the terms "complementary", "fully complementary" and "substantially complementary" can be used with respect to base pairing between the sense and antisense strands of dsRNA or between the antisense strand of a double-stranded RNAi agent and a target sequence, as understood from the context in which they are used.

[0125] 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 the mRNA of interest (e.g., the mRNA encoding KHK). For example, a polynucleotide is complementary to at least a portion of KHK mRNA if its sequence is substantially complementary to a contiguous portion of the mRNA encoding the KHK gene.

[0126] Thus, in certain embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target KHK sequence. In another embodiment, the antisense polynucleotides disclosed herein are substantially complementary to the target KHK sequence and are at least about 80% complementary, for example at least 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary or 100% complementary over a contiguous nucleotide sequence corresponding to a region of any one of SEQ ID NOs: 1, 3, 5, 7, 9 and 11, preferably SEQ ID NOs: 1, 3 and 5, or a fragment of any one of SEQ ID NOs: 1, 3, 5, 7, 9 and 11, preferably SEQ ID NOs: 1, 3 and 5, over its entire length.

[0127] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, which is complementary to a target KHK sequence and includes a sense strand nucleotide sequence from either Table 3 or Table 5, or a fragment of a sense strand from either Table 3 or Table 5, and a contiguous nucleotide sequence over its entire length that is at least about 80% complementary, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% complementary.

[0128] In one embodiment, the iRNA of the present invention comprises an antisense strand substantially complementary to a target KHK sequence, and includes a corresponding region of one of the nucleotide sequences of the antisense strands in Table 3 or Table 5, or a fragment of one of the antisense strands in Table 3 or Table 5, and a contiguous nucleotide sequence that is at least about 80% complementary, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary or about 100% complementary over its entire length.

[0129] Generally, the majority of nucleotides in each strand are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, “iRNA” may contain ribonucleotides with chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any such modification, such as those used in dsRNA molecules, is encompassed by “iRNA” for the purposes of this specification and the claims.

[0130] 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 target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to the sequence in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base-pairing with the mRNA and physically interfering with the translation mechanism (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may contain a sequence that is at least about 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0131] As used herein, the phrase “bring cells into contact with iRNA” includes the step of bringing cells into contact by any possible means. The step of bringing cells into contact with iRNA includes the step of bringing cells into contact with iRNA in vitro or the step of bringing cells into contact with iRNA in vivo. Contact may be direct or indirect. For example, iRNA may be brought into physical contact with cells by performing the method individually, or iRNA may be placed in a situation where it can be brought into contact with cells at a later date.

[0132] The process of contacting cells in vitro may be carried out, for example, by incubating cells with iRNA. The process of contacting cells in vivo may be carried out, for example, by injecting iRNA into or near the tissue where cells are present, or by injecting iRNA into another region, for example, the bloodstream or subcutaneous space, so that the iRNA reaches the tissue where the cells to be contacted are present after contact. For example, the iRNA may contain and / or be bound to a ligand that recruits the iRNA to a site of interest, for example, the liver, such as GalNAc3. It is also possible to combine in vitro and in vivo contact methods. For example, cells may be transplanted into a subject after being contacted with iRNA in vitro.

[0133] In one embodiment, the step of contacting cells with iRNA includes the step of “introducing” or “delivering iRNA into cells” by promoting or performing uptake or absorption into the cells. Absorption or uptake of iRNA may occur by unaided diffusive or active cellular processes, or by auxiliary drugs or devices. Introduction of iRNA into cells may be in vitro and / or in vivo. For example, in the case of in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be carried out by β-glucan delivery systems as described in U.S. Patent No. 5,032,401 and No. 5,607,677 and U.S. Patent Application Publication 2005 / 0281781, all of which are incorporated herein by reference. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further methods are described later in this specification and / or are known in the art.

[0134] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing a lipid layer that encapsulates a pharmaceutically effective molecule, such as a nucleic acid molecule (e.g., iRNA or a plasmid on which iRNA is transcribed). LNPs are described in U.S. Patents No. 6,858,225, No. 6,815,432, No. 8,158,601 and No. 8,058,069, all of which are incorporated herein by reference, for example.

[0135] As used herein, “Subject” is an animal, such as a primate (human, non-human primates, e.g., monkeys and chimpanzees), a mammal (including cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses and whales), or a bird (e.g., a duck or a goose) that expresses the target gene endogenously or exogenously. In some embodiments, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder or condition for which a reduction in the expression or replication of the target gene would be beneficial as described herein; a human being at risk for a disease, disorder or condition for which a reduction in KHK gene expression would be beneficial; a human being having a disease, disorder or condition for which a reduction in KHK gene expression would be beneficial; or a human being being treated for a disease, disorder or condition for which a reduction in KHK gene expression would be beneficial. In some embodiments, the subject is a human female. In other embodiments, the subject is a human male.

[0136] As used herein, the terms “to treat” or “to cure” include, but are not limited to, beneficial or desired outcomes, such as the alleviation or improvement of one or more symptoms or conditions associated with high KHK gene expression or high KHK protein production, particularly high KHK gene expression or high KHK protein production. “Treatment” may also mean extending survival compared to the survival expected in the event of no treatment.

[0137] In the context of a subject, the term “reduction” in relation to the level of KHK gene expression or KHK protein production, or to a disease marker or symptom, refers to a statistically significant reduction in such levels. A reduction could be, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the detection level of a detection method. In one embodiment, the expression of the target is reduced to a level that is normalized, i.e., within the normal range for an individual without the disorder, for example, within the normal range for body weight, blood pressure, or serum lipid levels. As used herein, “reduction” in a subject can refer to a reduction in gene expression or protein production in the cells of the subject, but it is not necessary to reduce expression in all cells or tissues of the subject. For example, as used herein, a reduction in a subject could include a reduction in gene expression or protein production in the liver of the subject.

[0138] The term "reduction" can also be used in relation to normalizing the symptoms of a disease or condition, including reducing the difference in range from a range in a subject with a KHK-related disease to a range in a normal subject without a KHK-related disease, or to the normal range in a subject without a KHK-related disease. For example, if a subject with a normal weight of 70 kg weighs 90 kg (20 kg overweight) before treatment and 80 kg (10 kg overweight) after treatment, the subject's weight has decreased by 50% (10 / 20 × 100%) relative to their normal weight. Similarly, if a woman's HDL level rises from 50 mg / dL (low) to 57 mg / dL, and the normal level is 60 mg / dL, the difference between the subject's pre-treatment level and the normal level has decreased by 70% (the difference between the subject's level and the normal level has decreased from 10 mg / dL to 7 mg / dL, 7 / 10 × 100%). As used herein, “normal” is considered the upper limit of normal when a disease is associated with a high value as a symptom. When a disease is associated with a low value as a symptom, “normal” is considered the lower limit of normal.

[0139] As used herein, “prevention” or “prevention” means, when used for a disease, disorder, or condition that benefits from reduced KHK gene expression or KHK protein production, a reduction in the likelihood of the subject developing symptoms associated with the said disease, disorder, or condition, such as symptoms or symptoms related to KHK gene expression, KHK activity, or high fructose metabolism. Without limiting the mechanism, KHK-catalyzed fructose phosphorylation is known to lead to uric acid production, unregulated by feedback inhibition resulting in ATP and intracellular phosphate depletion and increased AMP levels, by forming fructose-1-phosphate. Furthermore, fructose-1-phosphate is metabolized to glyceraldehyde, which is supplied to the citrate cycle, increasing the production of acetyl-CoA-stimulated fatty acid synthesis. Diseases and conditions associated with high uric acid and fatty acid synthesis include, for example, liver diseases (e.g., fatty liver, steatohepatitis including non-alcoholic steatohepatitis (NASH)), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low HDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance not related to an immune response to insulin, type II diabetes), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction), kidney diseases (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, chronic kidney disease), metabolic syndrome, lipid deposition or dysfunction diseases (e.g., adipocyte dysfunction, visceral fat deposition, obesity), high uric acid diseases (e.g., hyperuricemia, gout), and eating disorders such as excessive sugar cravings. Effective prevention is considered to be the prevention of the onset of a disease, disorder, or symptom, or the reduction of the onset of symptoms or comorbidities associated with such disease, disorder, or symptom (for example, by at least about 10% on a clinically recognized scale for the disease or disorder), or the delay of the onset of symptoms, symptoms, or disease progression for days, weeks, months, or years.

[0140] As used herein, the terms “ketohexokinase-related disease” or “KHK-related disorder” refer to a disease or disorder caused by or related to KHK gene expression or KHK protein production. The term “KHK-related disorder” encompasses diseases, disorders or conditions that benefit from reduced KHK gene expression, replication, or protein activity. Non-limiting examples of KHK-related disorders include, for example, liver diseases (e.g., fatty liver, steatohepatitis, e.g., non-alcoholic steatohepatitis (NASH)), dyslipidemia (e.g., hyperlipidemia, high LDL cholesterol, low LDL cholesterol, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance not related to an immune response to insulin, type II diabetes), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction), kidney diseases (e.g., acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, chronic kidney disease), metabolic syndrome, lipid deposition or dysfunction disorders (e.g., adipocyte dysfunction, visceral fat deposition, obesity, etc.), uric acid elevation disorders (hyperuricemia, gout, etc.), and eating disorders such as excessive sugar cravings. Further details regarding the symptoms and manifestations of various diseases or conditions are provided herein and are well known in the art.

[0141] In one embodiment, KHK-related diseases are associated with high uric acid levels (e.g., hyperuricemia, gout).

[0142] In one embodiment, KHK-related diseases are associated with high lipid levels (e.g., fatty liver, steatohepatitis including non-alcoholic steatohepatitis (NASH), and dyslipidemia).

[0143] As used herein, “therapeutic dose” is intended to include an amount of iRNA sufficient to treat a KHK-related disease when administered to a subject with the disease (for example, by reducing, alleviating, or maintaining one or more symptoms of the disease or its associated comorbidities). “Therapeutic dose” may vary depending on the method of iRNA administration, the disease and its severity, and the subject being treated, including their medical history, age, weight, family history, genetic makeup, stage of the KHK gene expression-mediated pathogenesis, the type of prior or concomitant treatment, if any, and other individual characteristics.

[0144] As used herein, “a prophylactically effective dose” is intended to contain an amount of iRNA sufficient to prevent or delay the onset or progression of the disease or one or more symptoms of the disease when administered to a subject who has not yet (or is not currently) exhibiting symptoms of the disease but is predisposed to developing the disease. “A prophylactically effective dose” may vary depending on the method of iRNA administration, the risk of developing the disease, and the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other individual characteristics.

[0145] The term "therapeutically effective dose" or "prophylactically effective dose" also includes the amount of iRNA that produces the desired local or systemic effect, which is a reasonable benefit / risk ratio applicable to any treatment. The iRNA used in the method of the present invention may be administered in an amount sufficient to obtain a reasonable benefit / risk ratio applicable to such treatment.

[0146] The phrase "pharmaceutically acceptable" is used herein to mean a compound, material, composition or dosage form suitable for use in contact with the tissues of human and animal subjects within reasonable medical judgment, commensurate with a reasonable benefit-risk ratio, without excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0147] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle involved in transporting or delivering a compound to a subject from one organ or part of the body to another organ or part of the body, such as liquid or solid fillers, diluents, excipients, manufacturing aids (e.g., lubricants, talc, magnesium stearate, calcium stearate or zinc stearate or stearic acid), or solvent encapsulating materials. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) celluloses such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, and their derivatives; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, 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; (10) propylene glycol and other compounds (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) Distilled water from which pyrogenic substances have been removed; (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, suitable substances used in pharmaceutical formulations.

[0148] As used herein, the term “sample” includes similar bodily fluids, cells, or tissues isolated from a subject, as well as aggregates of bodily fluids, cells, or tissues present in the subject. Examples of biological bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples obtained from tissues, organs, or local areas. For example, a sample may be obtained from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In one embodiment, a sample may be obtained from the liver (e.g., the whole liver or a specific part of the liver, or a specific type of cell in the liver, e.g., hepatocytes). In one embodiment, “sample obtained from subject” refers to urine obtained from the subject. In one embodiment, “sample obtained from subject” refers to blood obtained from the subject (which can be readily converted to plasma or serum).

[0149] II. The iRNA of the present invention The present invention provides an iRNA that inhibits the expression of the KHK gene. In a preferred embodiment, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the KHK gene in cells in subjects susceptible to KHK-related diseases, such as cells in mammals such as humans. The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during KHK gene expression. The complementary region is about 30 nucleotides or less in length (for example, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). When iRNA comes into contact with cells expressing the KHK gene, it inhibits the expression of the KHK gene (e.g., human, primate, non-primate, or avian KHK gene) by at least about 20% when assayed by, for example, PCR or branched DNA (bDNA) based methods, or protein-based methods (e.g., by immunofluorescence analysis using Western blotting or flow cytometry techniques). In preferred embodiments, inhibition of expression is determined by the qPCR method shown in Example 2, preferably by delivering the iRNA to a cell line at a concentration of 10 nM in a cell line matched to the appropriate species, using the method described herein.

[0150] dsRNA contains 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 (the antisense strand) contains a complementary region that is substantially, generally, fully complementary to the target sequence. The target sequence can be obtained from the sequence of mRNA formed during the expression of the KHK gene. The other strand (the sense strand) contains 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 in this specification and as known in the art, the complementary sequence of the dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, rather than on separate oligonucleotides.

[0151] Generally, a double-stranded structure has a length of 15-30 base pairs, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19- The lengths are 29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths within the above ranges and lengths are also considered to be part of the present invention.

[0152] Similarly, the complementary region of the target sequence has a nucleotide length of 15-30, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, The nucleotide lengths are 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22. Intermediate ranges and lengths within the above ranges and lengths are also considered to be part of the present invention.

[0153] In one embodiment, the dsRNA is approximately 15–23 nucleotides long, or approximately 25–30 nucleotides long. Generally, dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than approximately 21–23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is, in most cases, part of a larger RNA molecule (often an mRNA molecule). Where applicable, the "part" of the mRNA target is a contiguous sequence of mRNA targets long enough to exist as a substrate for RNAi-specific cleavage (i.e., cleavage via the RISC pathway).

[0154] The double-stranded region is the primary functional part of dsRNA, for example, approximately 9 to approximately 36 base pairs, for example, approximately 10 to 36, 11 to 36, 12 to 36, 13 to 36, 14 to 36, 15 to 36, 9 to 35, 10 to 35, 11 to 35, 12 to 35, 13 to 35, 14 to 35, 15 to 35, 9 to 34, 10 to 34, 11 to 34, 12 to 34, 13 to 34, 14 to 34, 15 to 34, 9 ~33, 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, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19 Those skilled in the art will also recognize that the base pairs are ~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. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs to the extent that it is processed into a functional double-stranded region of, for example, 15~30 base pairs that targets the desired RNA for cleavage is a dsRNA. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a natural miRNA. In another embodiment, iRNA agents useful for targeting KHK expression are not generated in target cells by cleaving larger dsRNAs.

[0155] The dsRNAs described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may comprise or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. Overhangs may be located on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0156] In some embodiments, overhangs on the sense strand, the antisense strand, or both strands may be longer than 10 nucleotides, for example, longer than 10–30 nucleotides, 10–25 nucleotides, 10–20 nucleotides, or 10–15 nucleotides. In some embodiments, the elongated overhang is located on the double-stranded sense strand. In certain embodiments, the elongated overhang is located at the 3' end of the double-stranded sense strand. In some embodiments, the elongated overhang is located at the 5' end of the double-stranded sense strand. In some embodiments, the elongated overhang is located on the double-stranded antisense strand. In some embodiments, the elongated overhang is located at the 3' end of the double-stranded antisense strand. In some embodiments, the elongated overhang is located at the 5' end of the double-stranded antisense strand. In some embodiments, one or more nucleotides in the elongated overhang are substituted with nucleoside thiophosphates.

[0157] dsRNA can be synthesized, for example, using an automated DNA synthesizer (such as one commercially available from Biosearch, Applied Biosystems, Inc.) by standard methods known in the art, as will be described further.

[0158] The double-stranded RNAi compounds of the present invention can be produced using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are produced separately. Next, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides can be readily prepared. Similarly, the single-stranded oligonucleotides of the present invention can be produced using solution-phase or solid-phase organic synthesis or both.

[0159] 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 Tables 3 and 5, and the corresponding antisense strand of the sense strand is selected from the group of sequences shown in Tables 3 and 5. 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 mRNA produced during KHK gene expression. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, where one oligonucleotide is represented as the sense strand in Table 3 or 5, and the second oligonucleotide is represented as the corresponding antisense strand of the sense strand in Table 3 or 5. 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.

[0160] Although the sequences in Table 3 are not listed as modified or conjugated sequences, it will be understood that the RNA of the iRNA of the present invention, for example, the dsRNA of the present invention, may include either a modified or conjugated sequence listed in Table 3, or an unmodified or unconjugated sequence listed in Table 5. In other words, the present invention includes unmodified, unconjugated, modified and / or conjugated dsRNAs listed in Tables 3 and 5, as described herein.

[0161] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of approximately 20–23 base pairs, for example, 21 base pairs, have been shown to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, the properties of the oligonucleotide sequences shown in one of Tables 3 and 5 may cause the dsRNAs described herein to contain at least one strand of at least 21 nucleotides in length. It can be naturally predicted that shorter double-stranded structures having one of the sequences in Tables 3 and 5 with a very small number of nucleotides subtracted from one or both ends may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotide sequences, obtained from one of the sequences shown in Tables 3 and 5, and having an ability to inhibit KHK gene expression that differs from that of dsRNAs containing the complete sequence by approximately 5, 10, 15, 20, 25, or 30% or less, are considered to be within the scope of the present invention.

[0162] Furthermore, the RNAs shown in Tables 3 and 5 identify sites in the KHK transcript that are susceptible to RISC-mediated cleavage. Therefore, the present invention further addresses iRNAs that target one or fewer of these sites. As used herein, an iRNA can be said to target a specific site within the RNA transcript if it induces cleavage of the transcript at any location within that particular site. Such an iRNA would generally contain at least about 15 consecutive nucleotides from one of the sequences shown in Tables 3 and 5, bound to a further nucleotide sequence obtained from a region adjacent to a selected sequence in the KHK gene.

[0163] Target sequences are generally about 15–30 nucleotides long, but the suitability of specific sequences within this range to guide the cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for identifying the optimal target sequence for any given gene target, an empirical approach can also be taken, which involves placing a “window” or “mask” of a given size (21 nucleotides as an example) literally or figuratively (including, e.g., in silico) on the target RNA sequence to identify sequences that fall within a size range that can function as a target sequence. Subsequent potential target sequences can be identified by gradually moving the sequence “window” upstream or downstream of one nucleotide from the initial target sequence location until a complete set of possible sequences has been identified for any given target size of choice. This process, along with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify the optimally functioning sequence, can identify the RNA sequence that best mediates the inhibition of target gene expression when targeted with an iRNA agent. Therefore, while the identified sequences, for example, those in Tables 3 and 5, represent effective target sequences, further optimization of inhibition efficiency can be achieved by gradually "shifting the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0164] Furthermore, for any sequence identified in Tables 3 and 5, for example, further optimization may be achieved by systematically adding or removing nucleotides to generate longer or shorter sequences, and by testing the resulting sequences by shifting the longer or shorter size window above or below the target RNA from its position. Also, in inhibitory assays known in the art and / or described herein, the efficiency of inhibition may be further improved by combining this method for generating novel candidate targets with testing the efficacy of iRNAs based on those target sequences. Moreover, such optimized sequences may be modified, for example, by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or by other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., increased serum stability or circulating half-life, increased thermal stability, improved membrane permeable delivery, targeting to specific locations or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes).

[0165] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch region is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is confined to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA agent, the strand complementary to the KHK gene region generally contains no mismatches in the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an iRNA containing a mismatch with the target sequence is effective in inhibiting KHK gene expression. Considering the effectiveness of an iRNA with a mismatch in inhibiting KHK gene expression is particularly important when it is known that a specific complementary region in the KHK gene has polymorphic sequence variation within the population.

[0166] II. Modified iRNA of the present invention In one embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified and does not contain, for example, any chemical modifications and / or conjugates known in the art and described herein. In another embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to improve stability or other beneficial properties. In one embodiment of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In another embodiment of the present invention, all of the nucleotides of the iRNA are modified, or substantially all of the nucleotides of the iRNA are modified, i.e., five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer unmodified nucleotides are present in the iRNA chain.

[0167] Nucleic acids relating to the present invention may be synthesized or modified by methods well established in the art, such as those described herein by reference in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugate, reverse bond) or 3'-terminal modifications (conjugate, DNA nucleotide, reverse bond, etc.); base modifications, e.g., substitution with stable bases, unstable bases or bases that base-pair with a wide range of partners, base removal (non-basic nucleotide) or conjugate bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; or skeletal modifications, e.g., modification or substitution of phosphodiester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNA containing a modified skeleton or RNA that does not contain natural nucleoside bonds. RNA containing a modified skeleton is particularly useful if it does not have a phosphorus atom in its skeleton. For the purposes of this specification, and as is sometimes referred to in the art, modified RNAs that do not contain a phosphorus atom in their internucleoside backbone can also be considered as oligonucleosides. In one embodiment, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0168] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates and other alkylphosphonates (e.g., 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, boranophosphates with the usual 3'-5' linkage, their 2'-5' linked analogues, and those with inverted polarity where adjacent pairs of nucleoside units are linked at 3'-5'~5'-3' or 2'-5'~5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0169] Representative U.S. patents teaching the manufacture of the phosphorus-containing bond described above include, but are not limited to, U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; and No. 5,321 ,131 specification; 5,399,676 specification; 5,405,939 specification; 5,453,496 specification; 5,455,233 specification; 5,466,677 specification; 5,476,925 specification ; 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, Specification No. 361; Specification No. 5,625,050; Specification No. 6,028,188; Specification No. 6,124,445; Specification No. 6,160,109; Specification No. 6,169,170; Specification No. 6,172,209; Specification No. 6,239,265; Specification No. 6,277,603; Specification No. 6,326,199; Specification No. 6,346,614; Specification No. 6,444,423; Specification No. 6,531,590; Specification No. 6,534,6 Examples include Patent No. 39; Patent No. 6,608,035; Patent No. 6,683,167; Patent No. 6,858,715; Patent No. 6,867,294; Patent No. 6,878,805; Patent No. 7,015,315; Patent No. 7,041,816; Patent No. 7,273,933; Patent No. 7,321,029; and U.S. Reissue Patent No. RE39464, the entirety of which is incorporated herein by reference.

[0170] Modified RNA skeletons that do not contain a phosphorus atom internally have skeletons formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include those having morpholino bonds (partially formed from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others having mixed N, O, S, and CH2 constituent parts.

[0171] Representative U.S. patents teaching the manufacture of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patent No. 5,034,506; No. 5,166,315; No. 5,185,444; No. 5,214,134; No. 5,216,141; No. 5,235,033; No. 5,64,562; No. 5,264,564; No. 5,405,938; No. 5,434,257; No. 5,466,677; No. 5,470,967; No. 5, Examples include Specification No. 489,677; Specification No. 5,541,307; Specification No. 5,561,225; Specification No. 5,596,086; Specification No. 5,602,240; Specification No. 5,608,046; Specification No. 5,610,289; Specification No. 5,618,704; Specification No. 5,623,070; Specification No. 5,663,312; Specification No. 5,633,360; Specification No. 5,677,437; and Specification No. 5,677,439, all of which are incorporated herein by reference.

[0172] A suitable RNA mimetic is conceived for use in iRNA, where both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with novel groups. The base units are maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound, an RNA mimetic, that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, the entirety of which is incorporated herein by reference. Further PNA compounds suitable for use with the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0173] Some embodiments of the present invention include RNA having a phosphorothioate skeleton and oligonucleosides having a heteroatom skeleton, in particular including --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene(methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [wherein the natural phosphodiester skeleton is represented as --O--P--O--CH2--], and the amide skeleton of U.S. Patent No. 5,602,240. In some embodiments, the RNA described herein has the morpholino skeleton structure of U.S. Patent No. 5,034,506.

[0174] Modified RNA may also contain one or more substituted sugar moieties. iRNAs as described herein, for example, dsRNAs, may contain at the 2' position one of the following: OH;F;O-,S- or N-alkyl;O-,S- or N-alkenyl;O-,S- or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 It can be an alkenyl or alkinyl. An example of a suitable modification is O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n It contains CH3)2, where n and m are 1 to about 10. In another embodiment, dsRNA has C1-C at the 2' position. 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, insertion groups, groups that improve the pharmacodynamic properties of iRNA or enhance the pharmacokinetic properties of iRNA, and one of other substituents having similar properties. In one embodiment, the modification includes 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other examples of modifications are 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the following examples herein. Further examples of modifications include: 5'-Me-2'-F nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (both R and S isomers of these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0175] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions in the iRNA's RNA, particularly on the 3' terminal nucleotide or at the 3' and 5' positions of the sugar in 2'-5' linked dsRNA. iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; Nos. 5,118,800; Nos. 5,319,080; Nos. 5,359,044; Nos. 5,393,878; Nos. 5,446,137; Nos. 5,466,786; Nos. 5,514,785; Nos. 5,519,134; and Nos. 5,567,81 Examples include Specification No. 1; Specification No. 5,576,427; Specification No. 5,591,722; Specification No. 5,597,909; Specification No. 5,610,300; Specification No. 5,627,053; Specification No. 5,639,873; Specification No. 5,646,265; Specification No. 5,658,873; Specification No. 5,670,633; and Specification No. 5,700,920, some of which are owned by the same person as the present application. All the contents of each of these are incorporated herein by reference.

[0176] iRNAs may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, and 5-uracil. This includes other synthetic and native nucleic acid bases such as (pseudracil), 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-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, which are also disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. The 5-methylcytosine substituent has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), which is a typical base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0177] Representative U.S. patents teaching the manufacture of the above-mentioned modified nucleic acid bases and some other modified nucleic acid bases include, but are not limited to, 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; and 5,594,1 Examples include Specification No. 21, No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; No. 6,166,197; No. 6,222,025; No. 6,235,887; No. 6,380,368; No. 6,528,640; No. 6,639,062; No. 6,617,438; No. 7,045,610; No. 7,427,672; and No. 7,495,088, all of which are incorporated herein by reference.

[0178] The RNA in iRNA can also be modified to include one or more lock nucleic acids (LNAs). A lock nucleic acid is a nucleotide with a modified ribose moiety, which contains additional crosslinks connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the structural configuration of the 3' end. Adding lock nucleic acids 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).

[0179] In one embodiment, the iRNA of the present invention comprises one or more monomers of nucleotides that are UNA (unlocked nucleic acid) nucleotides. UNA is an acyclic unlocked nucleic acid in which any sugar bond has been removed to form an unlocked "sugar" residue. In one example, UNA also comprises monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon bond, which is a covalent bond between C1' and C4') has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon bond, which is a covalent bond between C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039; incorporated herein by attribution).

[0180] Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, all of which are incorporated herein by reference.

[0181] The RNA of iRNA can be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge between two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge that connects two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In one embodiment, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in one embodiment, the agent of the present invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety, the ribose moiety including an additional bridge that connects the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose in the configuration of the 3'-terminal structure. The addition of locking nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between 4' and 2' ribosyl ring atoms. In one embodiment, the antisense polynucleotide agent of the present invention may be 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 “restricted ethyl” or “cEt”) and 4'-CH(CH2OCH3)-O-2' (and their analogues; see, for example, U.S. Patent No. 7,399,845); 4' -C(CH3)(CH3)-O-2' (and its analogs; see, for example, U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and its analogs; see, for example, U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, for example, U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (where R is H, C is C. 1- C 12 Examples include alkyl groups or protecting groups (see, for example, U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J.Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and its analogues; see, for example, U.S. Patent No. 8,278,426). All of the contents of each of these are incorporated herein by reference.

[0182] Further representative U.S. patents and publications teaching the preparation of lock 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; and Examples include U.S. Patent Application Publication No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281, all of which are incorporated herein by reference.

[0183] For example, any of the above bicyclic nucleosides having one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see International Publication No. 99 / 14226).

[0184] The RNA of the iRNA may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a restricted nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S configuration referred herein to as “S-cEt”.

[0185] The iRNA of the present invention may also include one or more "conformally constrained nucleotides" ("CRNs"). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRNs fix the ribose ring to a stable configuration and increase the affinity for hybridization to mRNA. The linker is long enough to position the oxygen optimally for stability and affinity and reduces puckering of the ribose ring.

[0186] Representative publications teaching some of the above preparations of CRN include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication No. 2013 / 036868, the entire contents of which are incorporated herein by reference.

[0187] Potentially stable modifications to the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3”-phosphate, and reverse base dT (idT). Disclosure of these modifications can be found in PCT publication number International Publication No. 2011 / 005861.

[0188] Other nucleotide modifications of the iRNA of the present invention include 5' phosphates or 5' phosphate mimetic, for example, a 5' terminal phosphate or phosphate mimetic on the antisense strand of the iRNA. Suitable phosphate mimetics are disclosed, for example, in U.S. Patent Publication 2012 / 0157511, the entirety of which is incorporated herein by reference.

[0189] A. Modified iRNA containing the motif of the present invention In one embodiment of the present invention, examples of double-stranded iRNA agents of the present invention include agents having chemical modifications as disclosed in WO2013 / 075035 (the entire contents of which are incorporated herein by reference). WO2013 / 075035 provides three identical modification motifs on three consecutive nucleotides in the sense strand and / or antisense strand of the dsRNAi agent, particularly at or near the cleavage site. In one embodiment, the sense strand and antisense strand of the dsRNAi agent may be modified or completely modified. The introduction of these motifs disrupts the modification pattern of the sense strand or antisense strand, if present. The DsRNAi agent may optionally be conjugated with a GalNAc derivative ligand on the sense strand, for example.

[0190] More specifically, gene silencing activity of the dsRNAi agent was observed when the sense and antisense strands of the double-stranded RNAi agent were completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0191] Accordingly, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the KHK gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may independently have a nucleotide length of 12 to 30. For example, each strand may independently have a nucleotide length of 14 to 30, 17 to 30, 25 to 30, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, or 21 to 23.

[0192] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "dsRNAi agent." The double-stranded region of the dsRNAi agent may be 12 to 30 nucleotide pairs long. For example, the double-stranded region may be 14 to 30 nucleotide pairs long, 17 to 30 nucleotide pairs long, 27 to 30 nucleotide pairs long, 17 to 23 nucleotide pairs long, 17 to 21 nucleotide pairs long, 17 to 19 nucleotide pairs long, 19 to 25 nucleotide pairs long, 19 to 23 nucleotide pairs long, 19 to 21 nucleotide pairs long, 21 to 25 nucleotide pairs long, or 21 to 23 nucleotide pairs long. In another example, the double-stranded region may be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide lengths.

[0193] In one embodiment, the dsRNAi agent may include one or more overhang regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may independently be 1 to 6 nucleotide lengths, e.g., 2 to 6 nucleotide lengths, 1 to 5 nucleotide lengths, 2 to 5 nucleotide lengths, 1 to 4 nucleotide lengths, 2 to 4 nucleotide lengths, 1 to 3 nucleotide lengths, 2 to 3 nucleotide lengths, or 1 to 2 nucleotide lengths. In one embodiment, the overhang region may include an extended overhang region as described above. The overhang may result from one strand being longer than the other, or from two strands of equal length being staggered. The overhang may form a mismatch with the target mRNA, or it may be complementary to or different from the targeted gene sequence. The first and second strands may also be joined, for example, by another base to form a hairpin, or by other non-base linkers.

[0194] In one embodiment, the nucleotides in the overhang region of the dsRNAi agent may be modified or unmodified nucleotides, each independently containing 2'-sugar modifications such as, but not limited to, 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT may be an overhang sequence for any end on any strand. The overhang may form a mismatch with the target mRNA, or the overhang may be complementary to the targeted gene sequence, or it may be a different sequence.

[0195] The 5'- or 3'-overhangs in the sense strand, antisense strand, or both strands of a dsRNAi agent may be phosphorylated. In one embodiment, the overhang region comprises two nucleotides having a phosphorothioate between them, where the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located in the antisense strand. In one embodiment, this 3'-overhang is located in the sense strand.

[0196] A dsRNAi agent may contain only one overhang that can enhance the interference activity of RNAi without affecting its overall stability. For example, the single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. The RNAi may 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 a dsRNAi agent has a nucleotide overhang at the 3' end and a blunt end at the 5' end. While we do not wish to be limited to theory, asymmetric blunt ends at the 5' end and 3' end overhangs of the antisense strand are favorable for introducing a guide strand into a RISC process.

[0197] In one embodiment, the dsRNAi agent is a double-stranded blunt-end with a length of 19 nucleotides, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides from the 5' end to positions 7, 8, and 9. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides from the 5' end to positions 11, 12, and 13.

[0198] In another embodiment, the dsRNAi agent is a double-stranded blunt-ended agent with a length of 20 nucleotides, wherein the sense strand contains at least one motif of three consecutive nucleotides with 2'-F modifications at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three consecutive nucleotides with 2'-O-methyl modifications at positions 11, 12, and 13 from the 5' end.

[0199] In yet another embodiment, the dsRNAi agent is a double-stranded blunt-end with a length of 21 nucleotides, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides from the 5' end to positions 9, 10, and 11. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides from the 5' end to positions 11, 12, and 13.

[0200] In one embodiment, the dsRNAi agent comprises 21 nucleotide sense strands and 23 nucleotide antisense strands, wherein the sense strands include at least one motif of three 2'-F modifications on three consecutive nucleotides from the 5' end to positions 9, 10, and 11; and the antisense strands include at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides from the 5' end to positions 11, 12, and 13, where one end of the iRNA agent is blunt, while the other end includes two nucleotide overhangs. Preferably, the two nucleotide overhangs are located at the 3' end of the antisense strand.

[0201] If two nucleotide overhangs are located at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide interbonds between the three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third nucleotide is a pairing nucleotide adjacent to the overhang nucleotides. In one embodiment, the RNAi agent further has two phosphorothioate nucleotide interbonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, all nucleotides in the sense and antisense strands of the dsRNAi 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 another motif). Optionally, the dsRNAi agent further comprises a ligand (preferably GalNAc3).

[0202] In one embodiment, a dsRNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long, with positions 1-23 of the first strand containing at least 8 ribonucleotides starting from the 5' terminal nucleotide (position 1); the antisense strand being 36-66 nucleotides long, with at least 8 ribonucleotides starting from the 3' terminal nucleotide, forming a double helix with positions paired with positions 1-23 of the sense strand; at least 3' terminal nucleotides of the antisense strand not paired with the sense strand, with up to 6 consecutive 3' terminal nucleotides not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; and 10-30 consecutive ribonucleotides not paired with the sense strand at the 5' end of the antisense strand. The sense strand contains nucleotides, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides; at least the 5' and 3' terminal nucleotides of the sense strand are bases that pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to the extent that the double-stranded nucleic acid reduces the expression of the target gene when introduced into mammalian cells; the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of the motifs is located at or near the cleavage site; the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0203] In one embodiment, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the dsRNAi agent comprises a first strand having a nucleotide length of at least 25 and no more than 29, and a second strand having a nucleotide length of no more than 30, containing at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at 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 to 4 nucleotides longer than the first strand at its 3' end, and the double-stranded region is at least 25 nucleotides long, and the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the second strand length to the extent that the RNAi agent reduces the expression of the target gene when introduced into mammalian cells, and the expression of the target gene in mammals is reduced by preferentially yielding an siRNA containing the 3' end of the second strand upon dicer cleavage of the dsRNAi agent. The dsRNAi agent may further contain a ligand, if desired.

[0204] In one embodiment, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications in three consecutive nucleotides, one of which is located at a cleavage site of the sense strand.

[0205] In one embodiment, the antisense strand of the dsRNAi agent may also include at least one motif of three identical modifications in three consecutive nucleotides, one of which is located at or near the cleavage site of the antisense strand.

[0206] In dsRNAi agents with double-stranded regions of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Therefore, 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 on the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand can also vary depending on the length of the double-stranded region of the dsRNAi agent from the 5' end.

[0207] The sense strand of a dsRNAi agent may contain at least one motif of three identical modifications in three consecutive nucleotides at the cleavage site of the strand; the antisense strand may have at least one motif of three identical modifications in three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand may have at least one nucleotide duplication in one of the three nucleotide motifs in the sense strand and one of the three nucleotide motifs in the antisense strand, i.e., at least one of the three nucleotides of the motif in the sense strand may be aligned to form a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may be duplicated, or all three nucleotides may be duplicated.

[0208] In one embodiment, the sense strand of a dsRNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides. The first motif may be located at or near the cleavage site of the strand, and the other motifs may be wing modifications. The term “wing modification” as used herein refers to a motif located in a different part of the strand, away from the motif at or near the cleavage site of the same strand. The wing modifications are adjacent to the first motif or separated by at least one or more nucleotides. If the motifs are directly adjacent to each other, their chemical structures are different from each other; if the motifs are separated by one or more nucleotides, their chemical structures may be the same or different. Two or more wing modifications may be present. For example, if two wing modifications are present, each wing modification may be located at one end relative to the first motif at or near the cleavage site, or on either side of the read motif.

[0209] Similar to the sense strand, the antisense strand of a dsRNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides, with at least one of the motifs located at or near the cleavage site of the strand. This antisense strand may contain one or more wing modifications in a sequence similar to the wing modifications that may be present in the sense strand.

[0210] In one embodiment, the wing modification in the sense or antisense strand of the dsRNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0211] In one embodiment, the wing modification in the sense or antisense strand of the dsRNAi agent typically does not contain the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0212] If the sense strand and antisense strand of a dsRNAi agent each contain at least one wing modification, the wing modifications may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0213] If the sense and antisense strands of a dsRNAi agent each contain at least two wing modifications, the sense and antisense strands may be aligned such that: two modifications from one strand are each located at one end of the double-stranded region and have one, two, or three nucleotide duplicates; two modifications from one strand are each located at the other end of the double-stranded region and have one, two, or three nucleotide duplicates; or two modifications from one strand are located on each side of the read motif and have one, two, or three nucleotide duplicates within the double-stranded region.

[0214] In one embodiment, all nucleotides in the sense and antisense strands of the dsRNAi agent, such as nucleotides that are part of a motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include changes to one or more unbound phosphate oxygens and / or bound phosphate oxygens; changes to components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; large-scale substitution of the phosphate moiety by a "dephospho" linker; modification or substitution of native bases; and substitution or modification of the ribose-phosphate backbone.

[0215] Because nucleic acids are polymers of subunits, many modifications, such as modifications to bases, or phosphate moieties, or unbound oxygen atoms of phosphate moieties, occur at repeating positions within the nucleic acid. In some cases, modifications may be present at all desired positions in the nucleic acid, but often this is not the case. For example, modifications may be present only at the 3' or 5' terminal position, or only in the terminal region, e.g., at a position on the terminal nucleotide, or only in the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may be present in the double-stranded region, the single-stranded region, or both. Modifications may be present only in the double-stranded region of a dsRNAi agent, or only in the single-stranded region of a dsRNAi agent. For example, phosphorothioate modifications at unbound oxygen positions may be present at only one or both ends, or only in the terminal region, e.g., at a position on the terminal nucleotide, or only in the last 2, 3, 4, 5, or 10 nucleotides of the strand, or in both the double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may also be phosphorylated.

[0216] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes (surrogates) in single-stranded overhangs, e.g., 5' or 3' overhangs, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of ribose sugars by modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modifications instead of ribosaccharides in nucleic acid bases, and modifications of phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0217] In one embodiment, each residue in the sense and antisense chains is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0218] At least two different modifications are typically present on the sense and antisense chains. These two modifications may be 2'-O-methyl or 2'-fluoro modifications or other modifications.

[0219] One reason, N a and / or N b This includes alternating pattern modifications. As used herein, the term “alternating motif” refers to a motif having one or more modifications, where each modification is alternately present on the nucleotides of a single chain. Alternating nucleotides may refer to one modification every other nucleotide, one modification every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motifs may be “ABABABABABAB···”, “AABBAABBAABB···”, “AABAABAABAAB···”, “AAABAAABAAAB···”, “AAABBBAAABBB···”, or “ABCABCABCABC···”.

[0220] The types of modifications included in alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, then the alternating pattern, i.e., the modifications on every other nucleotide, may be the same, but each of the sense or antisense strands may be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0221] In one embodiment, the dsRNAi agent of the present invention includes 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 may be such that the nucleotide modification groups in the sense strand correspond to different modification groups in the nucleotides of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double strand, the alternating motif in the sense strand may begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand may 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 may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from 5' to 3' of the strand within the double-stranded region, thereby resulting in a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

[0222] In one embodiment, the dsRNAi agent comprises a pattern of alternating 2'-O-methyl and 2'-F modifications in the sense strand, which initially has a shift relative to the pattern of alternating 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., a 2'-O-methyl modified nucleotide in the sense strand base-pairs with a 2'-F modified nucleotide in the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

[0223] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand disrupts the initial modification pattern present in the sense or antisense strand. This disruption of the modification pattern in the sense or antisense strand, achieved by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand, can enhance the gene silencing activity against the target gene.

[0224] In one embodiment, when three identical modification motifs on three consecutive nucleotides are introduced into any of the chains, the modifications of nucleotides adjacent to the motifs are different from the modifications of the motifs. For example, a portion of the sequence containing the motifs is "...N a YYYN b ..." where "Y" represents the modification of three identical modification motifs in three consecutive nucleotides, and "N a " and "N b " represents a modification of a nucleotide adjacent to the motif "YYY", which is different from the modification of Y, and N a and N b These can be the same or different modifications. Or, N a or N b This may or may not exist if a wing modifier is present.

[0225] iRNA may further contain at least one phosphorothioate or methylphosphonate internucleotide bond. The modification of the phosphorothioate or methylphosphonate internucleotide bond may be present on any nucleotide in the sense strand, antisense strand, or both strands, at any position on the strand. For example, the modification of the internucleotide bond may be present on all nucleotides in the sense strand and / or antisense strand; each modification of the internucleotide bond may be present in an alternating pattern in the sense strand and / or antisense strand; or the sense strand or antisense strand may contain modifications of both internucleotide bonds in an alternating pattern. The alternating pattern of the internucleotide bond modifications in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of the internucleotide bond modifications in the sense strand may have a shift relative to the alternating pattern of the internucleotide bond modifications in the antisense strand. In one embodiment, the double-stranded RNAi agent contains a 6-8 phosphorothioate internucleotide bond. In one embodiment, the antisense strand contains two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end, and the sense strand contains at least two phosphorothioate nucleotide interlinks at either its 5' or 3' end.

[0226] In one embodiment, the dsRNAi agent includes a modification of the overhang region with a phosphorothioate or methylphosphonate internucleotide bond. For example, the overhang region may include two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between them. The internucleotide bond modification may be formed to bind the overhang nucleotide to a terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4 or all of the overhang nucleotides may be bound by a phosphorothioate or methylphosphonate internucleotide bond, and optionally, there may be further phosphorothioate or methylphosphonate internucleotide bonds that bind the overhang nucleotide to a paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between three terminal nucleotides, two of which are overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. These three terminal nucleotides may be located at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0227] In one embodiment, two nucleotide overhangs are located at the 3' end of the antisense strand, with two phosphorothioate internucleotide bonds between the three terminal nucleotides, two of which are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. Optionally, the dsRNAi agent may further have two phosphorothioate internucleotide bonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0228] In one embodiment, the dsRNAi agent includes mismatches with the target, double-stranded mismatches, or combinations thereof. Mismatches may occur in overhang regions or double-stranded regions. Base pairs may be evaluated based on their tendency to promote dissociation or dissolution (e.g., with respect to the free energy of binding or dissociation of a particular pair; the simplest method is to examine each individual base pair, although 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; I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-standard or non-standard pairings (as described elsewhere in this specification), are preferred over standard (A:T, A:U, G:C) pairings; pairings containing universal bases are preferred over standard pairings.

[0229] In one embodiment, the dsRNAi agent comprises at least one of the first one, two, three, four, or five base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the groups A:U, G:U, and I:C, and a mismatch pair to facilitate the dissociation of the antisense strand at the 5' end of the double-stranded region, e.g., a non-standard or non-standard pairing or a pairing containing a universal base.

[0230] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0231] In one embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT), or the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). For example, there is a short sequence of deoxythymine nucleotides, and for example, there are two dT nucleotides at the 3' ends of the sense strand, the antisense strand, or both strands.

[0232] In one embodiment, the sense strand sequence is given by 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 independently either 0 or 1; p and q are independently between 0 and 6; each N a Each represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b This independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q This independently represents an overhanging nucleotide; Here, N b and Y do not have the same modifier; XXX, YYY, and ZZZ each independently represent a single motif of three identical modifications in three consecutive nucleotides. This can be shown by the following. Preferably, all YYY are 2'-F modified nucleotides.

[0233] One reason, N a or N b This includes alternating pattern modifications.

[0234] In one embodiment, the YYY motif is located at or near the sense strand cleavage site. For example, if the dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may be located at or near the sense strand cleavage site, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end (e.g., it may be located at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13).

[0235] 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 chain can 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).

[0236] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0237] If the sense chain is expressed as equation (Ic), then N bThis represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0238] If the sense chain is represented as expression (Id), then each N b This independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0239] Each of X, Y, and Z may be the same as or different from one another.

[0240] In another embodiment, when i is 0 and j is 0, the sense chain can be represented by the following equation: 5'n p -N a -YYY-N a -n q 3' (Ia).

[0241] If the sense chain is represented by equation (Ia), then each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0242] In one embodiment, the antisense strand sequence of RNAi is: Formula (II): 5'n q '-N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II) (In the formula, k and l are independently either 0 or 1; p' and q' are each independently between 0 and 6; each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p 'and n q ' independently represents an overhanging nucleotide; Here, N b 'and Y' do not have the same modifier; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It can be represented by:

[0243] One reason, N a 'or N b ' includes alternating pattern modifications.

[0244] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, if a dsRNAi agent has a double-stranded region of 17 to 23 nucleotides in length, the Y'Y'Y' motif may be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, from the 5' end, counting from the first paired nucleotide in the double-stranded region. Preferably, the Y'Y'Y' motif is located at positions 11, 12, and 13.

[0245] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0246] 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.

[0247] Therefore, the antisense chain is given 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) It can be represented by:

[0248] If the antisense chain is represented by equation (IIb), then N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0249] If the antisense chain is expressed as equation (IIc), then N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0250] If the antisense chain is expressed as equation (IId), then each Nb ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b It is 0, 1, 2, 3, 4, 5, or 6.

[0251] In another embodiment, when k is 0 and l is 0, the antisense chain can be represented by the following formula: 5'n p '-N a '-Y'Y'Y'-N a '-n q '3' (Ia).

[0252] If the antisense chain is represented by equation (IIa), then each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0253] Each of X', Y', and Z' may be the same as or different from one another.

[0254] Each nucleotide in the sense and antisense strands can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0255] In one embodiment, the sense strand of the dsRNAi agent may contain YYY motifs located at positions 9, 10, and 11 of the strand, starting from the first nucleotide from the 5' end, or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end; where Y represents a 2'-F modification. The sense strand may further contain XXX or ZZZ motifs as wing modifications at the opposite end of the double-stranded region; where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0256] In one embodiment, the antisense strand may contain a Y'Y'Y' motif located at positions 11, 12, and 13 of the strand, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end; where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0257] Each sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0258] Therefore, the dsRNAi agent for use in the method of the present invention may include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the iRNA double strand is given 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'np '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Here, each n p ',n p , n q 'and n q These independently represent overhang nucleotides, each of which may or may not be present; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications in three consecutive nucleotides. It is represented by [this].

[0259] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0260] An example of a sense strand and antisense strand combination that forms an iRNA double helix is ​​given by the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5' (IIId) These are some examples.

[0261] If a dsRNAi agent is represented by formula (IIIa), then each N a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0262] If a dsRNAi agent is represented by formula (IIIb), then each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0263] When a dsRNAi agent is represented by formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0264] When a dsRNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a , N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b Each of these independently includes alternating pattern modifications.

[0265] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), each of X, Y, and Z may be the same as or different from one another.

[0266] If a dsRNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), then at least one Y nucleotide may form a base pair with one of the Y' nucleotides; or at least two Y nucleotides may form base pairs with the corresponding Y' nucleotides; or all three Y nucleotides may form base pairs with the corresponding Y' nucleotides.

[0267] If a dsRNAi agent is represented by formula (IIIb) or (IIId), then at least one Z nucleotide may form a base pair with one of the Z' nucleotides; or at least two Z nucleotides may form base pairs with the corresponding Z' nucleotides; or all three Z nucleotides may form base pairs with the corresponding Z' nucleotides.

[0268] If a dsRNAi agent is represented by formula (IIIc) or (IIId), then at least one of its X nucleotides may form a base pair with one of its X' nucleotides; or at least two of its X nucleotides may form a base pair with the corresponding X' nucleotide; or all three of its X nucleotides may form a base pair with the corresponding X' nucleotide.

[0269] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0270] In one embodiment, if a dsRNAi agent is represented by formula (IIId), then N a The modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, if the RNAi agent is represented by formula (IIId), then N aThe modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is bound to an adjacent nucleotide via a phosphorothioate bond. In yet another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described below). In another embodiment, if the iRNA agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The sense strand is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker.

[0271] In one embodiment, if the dsRNAi agent is represented by formula (IIIa), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The nucleotide is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0272] In one embodiment, the dsRNAi agent is a multimer comprising at least two double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double strands being joined by a linker. The linker may be cleavable or incleavable. Optionally, the multimer further comprises a ligand. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0273] In one embodiment, the dsRNAi agent is a multimer comprising three, four, five, six or more double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double strands are linked by linkers. The linkers may be cleavable or incleavable. Optionally, the multimer further comprises ligands. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0274] In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are bound to each other at one or both of their 5' and 3' ends and optionally conjugated to a ligand. Each of the dsRNAi agents may target the same gene or two different genes; or each of the agents may target the same gene at two different target sites.

[0275] Various publications describe multimeric iRNAs that can be used in the methods of the present invention. Such publications include International Publication No. 2007 / 091269, U.S. Patent No. 7,858,769, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887, and International Publication No. 2011 / 031520, all of which are incorporated herein by reference.

[0276] As will be described in more detail below, iRNAs containing conjugations of one or more carbohydrate moieties can optimize one or more properties of the iRNA. Often, the carbohydrate moieties are bound to modified subunits of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA may be replaced by another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is bound. Ribonucleotide subunits in which the ribose sugar of a subunit is thus replaced are referred to herein as ribose-substituted modified subunits (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms are heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic system, or it may contain two or more rings, e.g., a fused ring. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

[0277] Ligands can be bound to polynucleotides via a carrier. The carrier comprises (i) at least one “skeletal attachment point,” preferably two “skeletal attachment points,” and (ii) at least one “tethering attachment point.” As used herein, “skeletal attachment point” refers to a functional group, e.g., a hydroxyl group, or more generally, a bond available and suitable for incorporating the carrier into the ribonucleic acid skeleton, e.g., a phosphate or modified phosphate (e.g., sulfur-containing) skeleton. A “tethering attachment point” (TAP) in some embodiments refers to a ring atom of a cyclic carrier linking a selected portion, e.g., a carbon atom or heteroatom (different from the atoms providing the skeletal attachment points). This portion may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Optionally, the selected portion is bound to the cyclic carrier by an intervening tether. Therefore, cyclic supports often contain functional groups, such as amino groups, or generally provide a bond suitable for the incorporation or tethering of another chemical component, such as a ligand, to the constituent ring.

[0278] The iRNA may be conjugated to a ligand via a carrier, which may be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0279] In a particular embodiment, the iRNA to be used in the method of the present invention is a drug selected from the group of drugs listed in Table 3 or 5. These drugs may further include ligands.

[0280] III. iRNA conjugated to a ligand Another modification of the iRNA of the present invention involves chemically attaching one or more ligands, parts, or conjugates to the iRNA that enhance the activity, cell distribution, or cell uptake (e.g., into cells) of the iRNA. These parts are not limited to, but include the cholesterol portion (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, for example, beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett.Examples of lipid moieties include, but are not limited to, those such as the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or the octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0281] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides enhanced affinity to selected targets (e.g., molecules, cells, or cell types), compartments (e.g., compartments of cells or organs), body tissues, organs, or regions, for example, compared to species without such ligands. Preferred ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0282] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be synthetic polymers, such as recombinants or synthetic molecules, including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-coglycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudo-peptide polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0283] Ligands may also include target groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, or antibodies that bind to specific cell types, such as kidney cells. Target groups may include thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, vitamin A, biotin, RGD peptides, or RGD peptide mimetic compounds.

[0284] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol), cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid Examples include dimethoxytrityl or phenoxazine, peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ tetraaza macrocyclic complexes), dinitrophenyl, HRP, or AP.

[0285] Ligands can be proteins, such as glycoproteins, or peptides, such as coligands, or antibodies, such as antibodies that bind to specific cell types, including hepatocytes. Ligands may also include hormones and hormone receptors. Ligands may also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species such as polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands may be, for example, lipopolysaccharides, activators of 38MAP kinase, or activators of NF-κB.

[0286] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton, for example, by disrupting the cellular microtubules, microfilaments, or intermediate filaments. Drugs may include, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0287] In some embodiments, ligands bound to iRNAs described herein serve as pharmacokinetic modifiers (PK modifiers). Examples of PK modifiers include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, and vitamins. Exemplary PK modifiers, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Many oligonucleotides containing phosphorothioate bonds are also known to bind to serum proteins; therefore, short oligonucleotides containing multiple phosphorothioate bonds in their backbone, such as oligonucleotides of approximately 5, 10, 15, or 20 bases, are also suitable 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.

[0288] The ligand-conjugate iRNAs of the present invention can be synthesized using oligonucleotides having reactive pendant functional groups, such as those derived from the binding of a binding molecule to the oligonucleotide (as described below). These reactive oligonucleotides may react directly with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands to which the binding group is attached.

[0289] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by well-known techniques relating to solid-phase synthesis. Apparatus for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means known in the art for such synthesis may be used additionally or alternatively. Similar techniques for preparing other oligonucleotides, such as phosphorothioates and alkylated derivatives, are also known.

[0290] In the ligand-conjugate iRNAs and ligand molecules having sequence-specific binding nucleosides of the present invention, oligonucleotides and oligonucleosides can be assembled in a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, nucleotide or nucleoside conjugate precursors already supporting binding groups, ligand-nucleotide or nucleoside conjugate precursors already supporting ligand molecules, or non-nucleoside ligand-containing building blocks.

[0291] When using nucleotide conjugate precursors that already have binding groups, typically, after the synthesis of sequence-specific binding nucleosides is complete, the ligand molecule reacts with the binding group to form ligand-conjugate oligonucleotides. In one embodiment, the oligonucleotides or binding nucleosides of the present invention are synthesized by an automated synthesizer using commercially available, standard and non-standard phosphoramidites conventionally used in oligonucleotide synthesis, in addition to phosphoramidites obtained from ligand-nucleoside conjugates.

[0292] A. Lipid conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues of the body, such as non-renal target tissues. For example, target tissues may be the liver, such as hepatic parenchymal cells. Other molecules that can bind to HSA may also be used as ligands. For example, naproxen or aspirin may be used. Lipid or lipid-based ligands may be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, or (c) modulate binding to serum proteins, such as HSA.

[0293] The binding of conjugates to target tissues can be inhibited, for example, controlled, using lipid-based ligands. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. Conjugates can be targeted to the kidneys using lipids or lipid-based ligands that bind more weakly to HSAs.

[0294] In one embodiment, a lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity such that the conjugate is preferably distributed to non-renal tissue. However, the affinity is preferably not so strong that the HSA-ligand binding may become irreversible.

[0295] In another embodiment, the lipid-based ligand binds weakly to or does not bind at all to the HSA so that the conjugate is preferably distributed to the kidney. Another portion targeting renal cells may also be used instead of, or in conjunction with, the lipid-based ligand.

[0296] In another embodiment, ligands are components taken up by target cells, such as proliferating cells, such as vitamins. These are particularly useful for treating, for example, undesirable cell proliferation, whether malignant or non-malignant, such as disorders characterized by cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells such as hepatocytes. HAS and low-density lipoprotein (LDL) are also included.

[0297] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, this agent is amphiphilic. Exemplary agents are peptides such as tat or Antennapedia. If this agent is a peptide, it may be modified, including the use of peptidyl mimes, inverted isomers, non-peptide or pseudopeptide bonds, and D-amino acids. The helical agent is preferably an α-helix agent, which preferably has lipophilic and oleophobic phases.

[0298] Ligands can be peptides or peptide mimes. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules that can fold into a defined three-dimensional structure similar to a natural peptide. Binding of peptides and peptide mimes to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptide mime portion may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0299] The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., mainly consisting of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In an alternative method, the peptide moiety may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). RFGF analogs containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 14)) can also be target moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. 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 function as delivery peptides. Peptides or peptide mimetic molecules can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84). (1991). Examples of peptides or peptide mimetic compounds linked to dsRNA agents via monomer units incorporated for cell targeting purposes include peptides such as arginine-glycine-aspartate (RGD)-peptides or RGD mimetic compounds. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion may have structural modifications, such as modifications that enhance stability or direct conformational properties. Any of the structural modifications described below may be used.

[0300] The RGD peptides used in the compositions and methods of the present invention may be linear or cyclic, and may be modified, for example, glycosylated or methylated, to facilitate targeting of specific tissues. RGD-containing peptides and peptide mimetic forms may include D-amino acids, as well as synthetic RGD mimetic forms. In addition to RGD, another moiety targeting an integrin ligand may be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0301] "Cell-permeable peptides" are capable of permeating cells, such as microbial cells like bacteria or fungal cells, or mammalian cells like human cells. Microbial cell-permeable peptides may be, for example, α-helix linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenecin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also include nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphipathic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0302] C. Carbohydrate Conjugate In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids as described herein, and the compositions are suitable for in vivo therapeutic use. As used herein, “carbohydrate” refers to a compound that is a carbohydrate itself, consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with each carbon atom bonded to an oxygen, nitrogen, or sulfur atom; or a compound having a carbohydrate moiety consisting of one or more monosaccharide units, each monosaccharide unit having at least six carbon atoms (which may be linear, branched, or cyclic), with each carbon atom bonded to an oxygen, nitrogen, or sulfur atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gum. Examples of specific monosaccharides include sugars with a HBV of 100% or higher (e.g., HBV, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., HBV, C6, C7, or C8).

[0303] In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is [ka] These include N-acetylgalactosamine.

[0304] In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is as follows: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0305] Another representative carbohydrate conjugate for use in the embodiments described herein is: [ka] (If one of X or Y is an oligonucleotide, the other is hydrogen.) This includes, but is not limited to, the following.

[0306] In one embodiment of the present invention, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a trivalent linker.

[0307] In one embodiment, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative conjugated to an iRNA agent. In another embodiment, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently conjugated to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0308] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule in which nucleotides are linked by an unbroken chain to form a hairpin loop consisting of multiple unpaired nucleotides between the 3' end of one strand and the 5' end of the other strand, each unpaired nucleotide in the hairpin loop may independently contain a GalNAc or GalNAc derivative linked via a monovalent linker.

[0309] In one embodiment, the carbohydrate conjugate further comprises one or more other ligands as described above, such as PK modulators or cell-permeable peptides, but is not limited to these.

[0310] Further carbohydrate conjugates suitable for use in the present invention include those described in International Publication Nos. 2014 / 179620 and 2014 / 179627 of the PCT Publications, the entire contents of which are incorporated herein by reference.

[0311] D. Linker In one embodiment, the conjugates or ligands described herein may be conjugated to iRNA oligonucleotides using a variety of linkers that may be cleavable or incleavable.

[0312] The term "linker" or "bonding group" refers to an organic part that connects two parts of a compound, for example, an organic part that covalently bonds two parts of a compound. Linkers are typically direct bonds or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or, but are not limited to, substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryls, heteroaryls, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls, alkenylheteroaryl Lukyl, alkenyl heteroaryl alkenyl, alkenyl heteroaryl alkynyl, alkynyl heteroaryl alkyl, alkynyl heteroaryl alkenyl, alkynyl heteroaryl alkynyl, alkyl heterocyclyl alkyl, alkyl heterocyclyl alkenyl, alkyl heterocyclyl alkynyl, alkenyl heterocyclyl alkyl, alkenyl heterocyclyl alkenyl, alkenyl heterocyclyl alkynyl, alkynyl heterocyclyl alkyl, alkynyl heterocyclyl alkenyl , comprising a chain of atoms such as alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl (where one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, or substituted or unsubstituted heterocyclic groups); where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker consists of approximately 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0313] The cleavable binding group is sufficiently stable outside the cell, but after entering the target cell, it is cleaved, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable binding group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the target blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum) within the target cell or under a first reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0314] Cleavable binding groups are sensitive to the presence of cleavage agents, such as pH, redox potential, or degradable molecules. Generally, cleavage agents are more widely present inside cells than in serum or blood, or are present at higher levels or activity. Examples of such degrading agents include redox agents selected for specific substrates or those without substrate specificity, such as intracellular oxidizers or reductases such as mercaptans that can degrade redox-cleavable binding groups by reduction; esterases; agents that can create endosomes or acidic environments, such as agents that lower the pH to below 5; and enzymes, peptidases (which may be substrate-specific) and phosphatases that can hydrolyze or degrade acid-cleavable binding groups by acting as general acids.

[0315] Cleavable binding groups, such as disulfide bonds, may be susceptible to pH. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of nearly 5.0. Some linkers, by possessing cleavable binding groups that are cleaved at a favorable pH, would release cationic lipids from ligands within the cell or into desired compartments of the cell.

[0316] Linkers may contain cleavable binding groups that can be cleaved by specific enzymes. The type of cleavable binding group incorporated into a linker can vary depending on the target cell. For example, a ligand targeting the liver may bind to cationic lipids via a linker containing an ester group. Because hepatocytes are rich in esterases, this linker will be cleaved more efficiently within hepatocytes compared to cell types that are less rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0317] Linkers containing peptide bonds can be used to target cell types that are rich in peptidases, such as hepatocytes and synovial cells.

[0318] Generally, the suitability of a candidate cleavable binding group can be evaluated by testing the ability of a degradation agent (or degradation condition) to cleave the candidate binding group. It is also desirable to test the ability of the candidate cleavable binding group to resist cleavage in blood or in contact with other non-target tissues. Specifically, the relative sensitivity to cleavage between a first and second condition can be determined, with the first condition selected to demonstrate cleavage within target cells, and the second condition selected to demonstrate cleavage in other tissues or body fluids, such as blood or serum. This evaluation can be performed in cell-free systems, intracellularly, in cell cultures, in organs or tissue cultures, or in whole animals. It may be useful to perform an initial evaluation in cell-free or cultured conditions and then confirm it with further evaluation in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster intracellularly (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).

[0319] i. Redox-cleavable bond groups In one embodiment, the cleavable binding group is a redox-cleavable binding group that is cleaved after reduction or oxidation. An example of a reductively cleavable binding group is a disulfide binding group (-SS-). To determine whether a candidate cleavable binding group is suitable as a “reductively cleavable binding group,” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent, one may look to the methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which mimics the rate of cleavage that may be observed in cells, for example, target cells. The candidate can also be evaluated under conditions selected to mimic the conditions of blood or serum, in one of which the candidate compound is cleaved by 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 intracellularly (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 candidate compounds can be determined using a standard enzyme kinetic assay under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0320] ii. Phosphate-based cleavable binding groups In another embodiment, the cleavable linker includes a phosphate-based cleavable binding group. The phosphate-based cleavable binding group is cleaved by an agent that breaks down or hydrolyzes the phosphate group. An example of an agent that cleaves the phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based binding groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0321] iii. Acid-cleavable bonding groups In another embodiment, the cleavable linker includes an acid-cleavable binding group. An acid-cleavable binding group is a binding group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable binding group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by a drug such as an enzyme that can act as a common acid. Within cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable binding groups. Examples of acid-cleavable binding groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may be represented by the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group (e.g., dimethylpentyl or t-butyl). These candidates can be evaluated using methods similar to those described above.

[0322] iv. Ester-based bonding groups In another embodiment, the cleavable linker includes an ester-based cleavable binding group. The ester-based cleavable binding group is cleaved by enzymes such as esterases and amylases in the cell. Examples of ester-based cleavable binding groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable binding group is represented by the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0323] v. Peptide-based cleavage groups In yet another embodiment, the cleavable linker includes a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. The peptide-based cleavable group is a peptide bond formed between amino acids, such as those that give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not contain an amide group (-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. The peptide-based cleavable group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, and does not include all amide functional groups. The peptide-based cleavable linking group is represented by the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0324] In one embodiment, the iRNA of the present invention is conjugated with a carbohydrate via a linker. Non-limiting examples of iRNA-carbohydrate conjugation with linkers of the compositions and methods of the present invention include, but are not limited to, the following: [ka] [ka] (In the formula, one of X or Y is an oligonucleotide, and the other is hydrogen.)

[0325] In one embodiment of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives linked via a divalent or trivalent branched linker.

[0326] In one embodiment, the dsRNA of the present invention is defined by formulas (XXXII) to (XXXV): [ka] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent a number from 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 of these is either nonexistent, or CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each of these is either nonexistent, an alkylene, or a substituted alkylene, where one or more methylene groups are one or more O, S, S(O), SO2, N(R) N ), C(R')=C(R”), C≡C or C(O) may interrupt or terminate the process; R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5CEach of these is independent of the others and either does not exist, or is represented by 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] It is a heterocycline; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C '' represents a ligand; that is, each independently is a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a [H is an amino acid side chain.] It is conjugated to a divalent or trivalent branched linker selected from the structural bases shown in any of the formulas. Trivalent conjugated GalNAc derivatives are RNAi agents for inhibiting the expression of target genes, e.g., formula (XXXV): [ka] (In the formula, L 5A , L 5B and L 5C (This represents monosaccharides such as GalNAc derivatives.) It is especially useful when used in conjunction with [another product / service].

[0327] Suitable divalent and trivalent branched binding groups for conjugating GalNAc derivatives include, but are not limited to, the structures described above as formulas II, VII, XI, X, and XIII.

[0328] Representative U.S. patents teaching 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; and 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; No. 4,789,737 ;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5 ,112,963;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5, No. 371,241; No. 5,391,723; No. 5,416,203; No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,567,810; 5,57 Nos. 4,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022 are cited, the entirety of which is incorporated herein by reference.

[0329] Not all positions of a given compound need to be uniformly modified; in fact, two or more of the above modifications can be incorporated into a single compound or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0330] It is not necessary for all positions of a given compound to be uniformly modified; in fact, two or more of the above modifications can be incorporated into a single compound or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0331] In the context of this specification, a “chimeric” iRNA compound or “chimeric” is an iRNA compound, preferably a dsRNAi agent, that contains two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region, where the RNA is modified to give the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity to a target nucleic acid. Another region of the iRNA may act as a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand in an RNA:DNA double-stranded DNA. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. Consequently, when chimeric dsRNAs are used, equivalent results are often obtained with shorter iRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of RNA targets can be conventionally detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0332] In some cases, the RNA of an iRNA can be modified with a non-ligand group. Many non-ligand molecules are conjugated to iRNAs to improve their activity, cell distribution, or cell uptake, and procedures for such conjugations can be found in the scientific literature. Such non-ligand moieties include, for example, 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, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantane acetate (Manoharan et al.These RNA conjugates include lipid moieties such as (Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugate protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino groups are then reacted with the conjugated molecule using a suitable coupling agent or activating reagent. The conjugate reaction can be performed using RNA still bound to a solid support or after cleavage of RNA in the solution phase. Purification of the RNA conjugate by HPLC usually yields a pure conjugate.

[0333] IV. Delivery of iRNA according to the present invention The iRNA of the present invention can be delivered to cells in a target, such as a human target (e.g., a target requiring an iRNA agent, such as a target with a disease, disorder, or condition related to KHK gene expression), in several different ways. For example, delivery may be carried out by bringing cells into contact with the iRNA of the present invention either in vitro or in vivo. In vivo delivery may also be carried out directly by administering a composition containing the iRNA, such as dsRNA, to the target. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode and lead to the expression of the iRNA. These alternatives are further described below.

[0334] In general, any method for delivering nucleic acid molecules (in vitro or in vivo) may be adapted for use with the iRNA of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, considerations for delivering the iRNA molecule 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, for example, by direct injection or transplantation into tissue, or by local administration of the formulation. Local administration to the treatment site can maximize the local concentration of the drug and limit the total dose of the administered iRNA molecule by limiting exposure of the drug to systemic tissues that may be adversely affected by the drug or where the drug may be degraded. Several studies have demonstrated successful knockdown of gene products when dsRNAi is administered locally. For example, intravitreal injection of VEGF dsRNA into cynomolgus monkeys (Tolentino, MJ, et al (2004) Retina 24:132-138) and subretinal injection into mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) both demonstrated prevention of neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and extended the lifespan 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, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and locally to the lungs by intranasal administration (Howard, KA., et al (2006) Mol. Ther. Success has also been demonstrated by (14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For systemic administration of iRNA for the treatment of disease, the RNA can be modified or delivered using a drug delivery system; both methods act to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or drug carriers can also enable the targeting of iRNA to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical binding to lipophilic groups such as cholesterol, which improves cellular uptake and prevents degradation. For example, when iRNA against ApoB conjugated to a lipophilic cholesterol portion was systemically administered to mice, knockdown of apoB mRNA was achieved in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).Conjugate of iRNA to aptamers has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO, et al (2006) Nat. Biotechnol. 24:1005-1015). In another embodiment, iRNA may be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems can facilitate the binding of negatively charged iRNA molecules and enhance interactions with negatively charged cell membranes, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can bind to iRNA or be induced to form vesicles or micelles that enclose the iRNA (see, e.g., Kim SH, et al (2008) Journal of Controlled Release 129(2):107-116). Vesicle or micelle formation further prevents the degradation of iRNA when administered systemically. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR, et al (2003) J. Mol. Biol 327:761-766; Verma, UN, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (SSorensen, DR., et al (2003), see above; Verma, UN, et al (2003), see above), oligofactamines, i.e., "solid nucleic acid lipid particles" (Zimmermann, TS, et al (2006) Nature 441:111-114), cardiolipin (Chien, PY, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), and polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. Examples include iRNAs (71659), Arg-Gly-Asp(RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, DA, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In one embodiment, iRNAs form a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins are found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in whole.

[0335] A. The iRNA of the present invention encoded by a vector iRNAs targeting the KHK 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; see Skillern, A et al., International PCT Publication No. 00 / 22113). Expression can be transient (approximately a few hours to several weeks) or persistent (several weeks to several 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 embedded or non-embedded vectors. Transgenes can also be constructed to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0336] Individual strands of iRNA can be transcribed from a promoter in an expression vector. If two separate strands are expressed to produce, for example, dsRNA, then two separate expression vectors can be co-introduced (e.g., by transfection or infection) into target cells. Alternatively, each individual strand of dsRNA can be transcribed by both promoters located on the same expression plasmid. In one embodiment, dsRNA is expressed as a reverse repeat polynucleotide joined by a linker polynucleotide sequence to have a stem-loop structure.

[0337] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for iRNA expression described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from many commercial sources. Such vectors are usually provided that contain restriction sites convenient for inserting the desired nucleic acid segment. iRNA expression vectors can be delivered by systemic administration, e.g., intravenous or intramuscular administration, by administration to target cells transplanted from the patient and then reintroduction into the patient, or by any other means that enable introduction into the desired target cells.

[0338] Viral vector systems that may be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including, but not limited to, lentivirus vectors and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, such as vaccinia virus vectors or avian pox, such as 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 be incorporated into the cell genome. The construct may optionally contain a viral sequence for transfection. Alternatively, the construct may be incorporated into an episomal replication-capable vector, such as EPV and EBV vectors. Constructs for the recombinant expression of iRNA generally require regulatory elements, such as promoters and enhancers, to ensure iRNA expression within target cells. Other embodiments of vectors and constructs considered are known in this field.

[0339] V. Pharmaceutical Composition 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 comprising the iRNA described herein and a pharmaceutically acceptable carrier is also provided herein. The iRNA-containing pharmaceutical composition is useful for treating diseases or disorders related to the expression or activity of the KHK gene. Such pharmaceutical compositions are formulated based on a delivery method. One example is a composition formulated for systemic administration via parenteral administration, for example, by subcutaneous (SC) or intravenous (IV) delivery. The pharmaceutical composition of the present invention may be administered in a dose sufficient to inhibit the expression of the KHK gene.

[0340] The pharmaceutical composition of the present invention can be administered in a dose sufficient to inhibit the expression of the KHK gene. Generally, appropriate doses of the iRNA of the present invention are in the range of about 0.001 to about 200.0 mg / kg body weight / day, usually about 1 to 50 mg / kg body weight / day. Typically, appropriate doses of the iRNA of the present invention are in the range of about 0.1 mg / kg to about 5.0 mg / kg, preferably about 0.3 mg / kg to about 3.0 mg / kg. Repeated-dose regimens may include administering therapeutic doses of iRNA regularly, for example, every other day or once a year. In some embodiments, iRNA is administered about once a month to about once a quarter (i.e., once every three months).

[0341] After the initial treatment regimen, the therapeutic agent may be administered less frequently. For example, after administration weekly or bi-weekly for three months, administration may be repeated monthly for six months, one year, or longer.

[0342] The pharmaceutical composition may be administered once daily, or the iRNA may be administered in two or three or more divided doses at appropriate intervals throughout the day, or further, by delivery via continuous infusion or sustained-release formulation. In this case, the amount of iRNA contained in each divided dose must be correspondingly smaller in order to achieve the total daily dose. The dose units may also be formulated for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of iRNA over a period of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites, and can therefore be used with the drug of the present invention. In this embodiment, the dose units comprise a corresponding multiple of the daily dose.

[0343] In another embodiment, since a single dose of the pharmaceutical composition can be continued for a long period, subsequent doses are administered at intervals of 3, 4, or 5 days or less, or at intervals of 1, 2, 3, or 4 weeks or less. In one embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once every two months.

[0344] Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or illness, treatment history, the subject's overall health or age, and other pre-existing diseases, may influence the dosage and time required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of the composition may comprise a single treatment or a series of treatments. The effective dosage and in vivo half-life for each iRNA encompassed by the present invention can be estimated using conventional methodologies or based on in vivo studies using suitable animal models known in the art. Suitable animal models for various diseases and conditions are provided herein.

[0345] The pharmaceutical composition of the present invention may be administered in several ways, depending on whether local or systemic treatment is required and the site to be treated. Administration may be local (e.g., by a transdermal patch), pulmonary administration by inhalation or blowing of powder or aerosol, such as by a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral administration. Parenteral administration may be intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; for example, subcutaneous administration by an implantable device; or, for example, intracranial administration by intraparenchymal, intrathecal or intraventricular administration.

[0346] iRNAs can be delivered in a manner that targets specific tissues (e.g., vascular endothelial cells).

[0347] Pharmaceutical compositions and formulations for topical or transdermal administration include transdermal patches, ointments, lotions, creams, gels, droplets, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., are required or may be desired. Covered condoms and gloves may also be useful. Suitable topical formulations include those in which the iRNA characterizing the present invention is a mixture with a topically delivered agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNAs characterizing the present invention can be encapsulated in liposomes or can form complexes with liposomes, particularly cationic liposomes. Alternatively, the iRNAs may be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaplate, tricaplate, monoolein, dilaurin, glyceryl 1-monocaplate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Examples include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof). Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0348] A. iRNA preparations 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 monolayer and multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains iRNA. The lipophilic material separates the aqueous interior from the aqueous exterior and usually does not contain the iRNA composition, but may contain it depending on the circumstances. Liposomes are useful for the transport and delivery of active ingredients to the site of action. Because the liposome membrane is structurally similar to that of biological membranes, when liposomes adhere to tissue, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the fusion of the liposome and the cell progresses, the aqueous substance inside containing the iRNA is delivered to the cell, where the iRNA can specifically bind to target RNA and mediate RNA interference. In some cases, liposomes can also be specifically targeted, for example, to direct iRNA towards a particular cell type.

[0349] Liposomes containing iRNA agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed together with the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholates, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Next, the iRNA agent preparation is added to the micelles containing the lipid component. The cationic groups in the lipids interact with the iRNA agent and condense around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain the liposomal formulation of the iRNA agent.

[0350] If necessary, a support compound to assist in condensation may be added during the condensation reaction, for example, by controlled addition. For example, the support compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). It is also possible to adjust the pH to assist in condensation.

[0351] A method for generating a stable polynucleotide delivery vehicle incorporating a polynucleotide / cationic lipid complex as a component of the delivery vehicle is further described, for example, in International Publication No. 96 / 37194, which is incorporated herein by reference, and all of its contents are incorporated herein by reference. Liposome formation is described in Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. 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. 1983; and Fukunaga, et al. This may also include one or more embodiments of the exemplary methods described in Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid assemblies of a suitable size for use as a delivery vehicle include sonication, freeze-thaw, and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50–200 nm) and relatively uniform assemblies are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily applicable for packaging iRNA preparations into liposomes.

[0352] Liposomes are broadly classified into two types. 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. Due to the acidic pH inside the endosomes, the liposomes rupture, and their contents are released into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0353] pH-sensitive or negatively charged liposomes capture nucleic acids rather than complex with them. Since both nucleic acids and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are taken up into the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to cell monolayers in cultures. Expression of exogenous genes has been detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0354] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions may be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, but anionic membrane-fused liposomes are primarily formed from dioleylphosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soy PC and egg PC. Other types are formed from mixtures of two or more phospholipids, phosphatidylcholine, and cholesterol.

[0355] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent No. 5,283,185; U.S. Patent No. 5,171,678; International Publication No. 94 / 00569; International Publication No. 93 / 24640; International Publication No. 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0356] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in drug delivery to the skin. Cyclosporine-A was delivered to the dermis of mouse skin using nonionic liposome formulations containing Novasome® I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome® II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results showed that such nonionic liposome systems are effective in promoting the deposition of cyclosporine-A into different layers of the skin (Hu et al. STPPharma. Sci., 1994, 4(6) 466).

[0357] Liposomes also include “stereostabilized” liposomes, and as used herein, this term refers to liposomes containing one or more specific lipids, the cyclic lifespan of which, when incorporated into the liposome, is increased compared to liposomes that do not contain such specific lipids. An example of a stereostabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome contains (A) monosialoganglioside G M1(B) These include those containing one or more glycolipids, or those derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not bound by any particular theory, in the art, with regard to sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivativeized lipids, the enhanced circulating half-life of these sterically stabilized liposomes is thought to be due to reduced intracellular uptake by the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0358] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) developed monosialoganglioside G to improve the blood half-life of liposomes. M1 The capabilities of galactocerebroside sulfate and phosphatidylinositol have been reported. These findings are detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924 (both by Allen et al.) describe (1) sphingomyelin and (2) ganglioside G M1 Alternatively, liposomes containing galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0359] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes are not as efficient at fusing with the cell membrane, but they can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0360] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can encapsulate a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect iRNA agents encapsulated within their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.)). Important considerations in the preparation of liposomal formulations are lipid surface charge, vesicle size, and the volume of water soluble substances in the liposomes.

[0361] Using N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a positively charged synthetic cationic lipid, it is possible to form small liposomes that spontaneously interact with nucleic acids and fuse with negatively charged lipids in the cell membrane of tissue culture cells, forming lipid-nucleic acid complexes capable of delivering iRNA agents (see, for example, Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; and U.S. Patent No. 4,897,355 for a description of its use with DOTMA and DNA).

[0362] 1,2-bis(oleyloxy)-3-(trimethylammonia)propane (DOTAP), a DOTMA analog, 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 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 complex thus prepared spontaneously attaches to negatively charged cell surfaces, fuses with the cell membrane, and efficiently delivers functional nucleic acids, for example, into 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 portion is linked by an ester rather than an ether linkage.

[0363] Other reported cationic lipid compounds include those conjugated to various functional groups, such as carboxyspermine conjugated to one of two lipids, and compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam®, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES") (see, for example, U.S. Patent No. 5,171,678).

[0364] Another cationic lipid conjugate includes a lipid derivative ("DC-Chol") of cholesterol formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for oligonucleotide delivery are described in International Publication No. 98 / 39359 and International Publication No. 96 / 37194.

[0365] Liposome formulations are particularly well-suited for topical administration, and they 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 drug at the desired target, and the ability to deliver iRNA agents to the skin. In some applications, liposomes are used to deliver iRNA agents to epidermal cells and to facilitate their penetration into dermal tissue, such as the skin. For example, liposomes can be applied topically. Local 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., Antivirus Research, 18, 1992, 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, RM and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, CY and Huang, L., Proc. See Natl. Acad. Sci. USA 84:7851-7855, 1987.

[0366] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in drug delivery 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.

[0367] iRNA-containing liposomes can be made into highly deformable shapes. Such deformability can allow 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 surface edge activators, usually surfactants, to a standard liposome composition. iRNA-containing transfersomes can be delivered to keratinocytes in the skin, for example, by subcutaneous infection. To pass through intact mammalian skin, the lipid vesicles must pass through a series of micropores, each having a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to their lipid properties, these transfersomes can be self-optimizing (e.g., adaptable to the shape of pores), self-repairing, often reach their targets without rupture, and often self-loading.

[0368] Other formulations suitable for the present invention are also described in WO2008 / 042973.

[0369] Transfersomes are another type of liposome, highly deformable lipid aggregates that are attractive candidates as drug delivery vehicles. Transfersomes can also be described as lipid droplets, which, due to their high deformability, can easily permeate smaller pores than droplets. Transfersomes are adaptable to the environment in which they are used, for example, self-optimal (adapting to the shape of pores in the skin), self-repairing, reaching their targets in large quantities without fragmentation, and often self-carrying. To construct transfersomes, surface-edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0370] Surfactants have found broad applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the numerous different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0371] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have found a wide range of applications in pharmaceutical and cosmetic products and can be used across a wide range of pH values. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Examples of nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0372] When a surfactant molecule retains a negative charge when dissolved or dispersed in water, it is classified as anionic. Anionic surfactants include carboxylates such as soap, acyl lactylates, acylamides of amino acids, sulfate esters 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 the anionic surfactant class are alkyl sulfates and soaps.

[0373] When a surfactant molecule retains a positive charge when dissolved or dispersed in water, it is classified as a cationic surfactant. Examples of cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most widely used members of this class.

[0374] A surfactant is classified as amphoteric if its molecule has the ability to possess either a positive or negative charge. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

[0375] The use of surfactants in pharmaceutical products, formulations, and emulsions has been outlined (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0376] iRNA for use in the method of the present invention may be provided as a micelle formulation. A “micelle” is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecule face inward and the hydrophilic portions are in contact with the surrounding aqueous phase. The reverse configuration exists when the environment is hydrophobic.

[0377] Mixed micelle formulations suitable for transcutaneous delivery include aqueous solutions of iRNA and alkali metals C8-C8. 22 It 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, trihydroxyoxocolanyglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogues, polydocanol alkyl ethers and their analogues, chenodeoxycholates, deoxycholates and mixtures thereof. The micelle-forming compounds may be added simultaneously with or after the addition of alkali metal alkyl sulfates. Mixed micelles are formed by substantially any kind of mixing of the components, but are formed by vigorous mixing to provide smaller sized micelles.

[0378] In one method, a first micelle composition containing RNAi 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 RNAi, an alkali metal alkyl sulfate, and at least one micelle-forming compound, and then adding the remaining micelle-forming compounds while vigorously mixing.

[0379] Phenol or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect it from bacterial growth. Alternatively, phenol or m-cresol may be added together with the micelle-forming components. An isotonic agent such as glycerin may also be added after the formation of the mixed micelle composition.

[0380] To deliver micelle formulations as a spray, the formulation can be placed in an aerosol dispenser, and the dispenser is filled with a propellant. Under pressurization, the propellant is in liquid form within the dispenser. The ratio of components is adjusted so that there is one aqueous phase and one propellant phase, i.e., one phase exists. If two phases exist, for example, a metering valve may be used to shake the dispenser before dispensing a portion of the contents. The dosage of the drug is dispensed from the metering valve in the form of a fine spray.

[0381] The propellant may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ethers, and diethyl ethers. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0382] The specific concentration of essential components can be determined by relatively simple experiments. For oral absorption, it is often desirable to increase the dosage to, for example, at least two or three times the dosage used for injection or administration via the gastrointestinal tract.

[0383] B. Lipid particles The iRNA of the present invention, for example, the dsRNAi agent, may be completely encapsulated in a lipid formulation, for example, in LNP, or may form other nucleic acid-lipid particles.

[0384] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs are extremely useful for systemic application because they have a long circulating lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include "pSPLPs," which contain encapsulated condensant-nucleic acid complexes, as shown in PCT Publication International Publication No. 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 non-toxic. In addition, when nucleic acids are present in the nucleic acid-lipid particles of the present invention, they are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent No. 5,976,567; U.S. Patent No. 5,981,501; U.S. Patent No. 6,534,484; U.S. Patent No. 6,586,410; U.S. Patent No. 6,815,432; U.S. Patent Publication No. 2010 / 0324120; and PCT International Publication No. 96 / 40964.

[0385] In one embodiment, the lipid-to-drug ratio (mass / mass ratio) (for example, the lipid-to-dsRNA ratio) may be within the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1. Ranges between the above ranges are also considered to be part of the present invention.

[0386] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcal Bamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogues It may also be (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazanegiyl)didodecane-2-ol (Tech G1) or a mixture thereof. Cationic lipids can constitute approximately 20 mol% to 50 mol%, or even 40 mol%, of the total lipids present in the particles.

[0387] In one embodiment, the compound 2,2-dirinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane may be used to prepare lipid-siRNA nanoparticles.

[0388] In one embodiment, the lipid-siRNA particles consist of 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (mol percent), with a particle size of 63.0 ± 20 nm and a siRNA / lipid ratio of 0.027.

[0389] Ionic / non-cationic lipids include, without limitation, distearoyl phosphatidylcholine (DSPC), dioleyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), and dioleyl-phosphatidylethanolamine. The anionic or neutral lipids may include 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Non-cationic lipids, when cholesterol is present, may be present in amounts of about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0390] Conjugate lipids that inhibit particle aggregation may include, for example, polyethylene glycol (PEG)-lipids containing, non-limitingly, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates may include, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (Ci8). The conjugate lipids that prevent particle aggregation may be present in the particles at a concentration of 0 mol% to approximately 20 mol% or 2 mol% of the total lipids present in the particles.

[0391] In one embodiment, the nucleic acid-lipid particles further contain, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particles, which is cholesterol.

[0392] In one embodiment, the lipidoid ND98·4HCl (MW 1487) (reference; U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) may be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Each stock solution in ethanol may be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-Ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-Ceramide C16 may then be combined, for example, in a molar ratio of 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., in sodium acetate (pH 5)) such that the final concentration of ethanol is approximately 35-45% and the final concentration of sodium acetate is approximately 100-300 mM. Lipid-dsRNA nanoparticles are usually formed spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., with a 100 nm shear) using a thermobarrel extruder, such as a Lipex extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate-buffered saline (PBS) at approximately pH 7, e.g., approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4.

[0393] [ka]

[0394] The LNP01 formulation is described, for example, in the brochure International Publication No. 2008 / 042973, which is incorporated herein by reference.

[0395] Further examples of lipid-dsRNA formulations are listed in Table 1. [Table 1-1] [Table 1-2] 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-distylylglycerol (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(l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. 2009 / 127060, filed on April 15, 2009, and are incorporated herein by reference.

[0396] Formulations containing XTC are described, for example, in International Application PCT / US2010 / 022614, filed on 29 January 2010, which is incorporated herein by reference.

[0397] Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed on June 10, 2010, all of which are incorporated herein by reference.

[0398] Formulations containing ALNY-100 are described, for example, in International Patent Application PCT / US09 / 63933, filed November 10, 2009, which are incorporated herein by reference.

[0399] Formulations containing C12-200 are described in WO2010 / 129709, which are incorporated herein by reference.

[0400] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, turbidiants or liquids in water or aqueous media, capsules, gel capsules, medicine bags, tablets or small tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desired. In one embodiment, the oral formulation is administered with one or more permeabilizing surfactants and chelating agents, containing the dsRNA characteristic of the present invention. Suitable surfactants include fatty acids 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, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of osmotic enhancers, such as fatty acids / salts combined with bile acids / salts, is used. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNAs characterizing the present invention can be delivered orally in granular form, including spray-dried particles or those complexed to form micro or nanoparticles.dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates; cationic gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pullulan, cellulose and starch. Preferred 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, DEA Examples include E-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginate and polyethylene glycol (PEG)). Oral formulations for dsRNA and preparations thereof are described in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 2003 / 0027780 and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

[0401] Compositions and formulations for parenteral, intraparenchymal (into the brain), subarachnoid, intraventricular, or intrahepatic administration include sterile aqueous solutions, which may contain buffers, diluents, and other suitable additives, such as, but are not limited to, osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0402] The pharmaceutical compositions of the present invention include, but are not limited to, liquid formulations, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but are not limited to, pre-formed liquid formulations, self-emulsifying solids, and self-emulsifying semi-solids. The formulations include those that target the liver when treating liver diseases such as hepatocarcinoma.

[0403] The pharmaceutical formulations of the present invention, which can be readily available in unit dosage forms, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of mixing the active ingredient with a pharmaceutical carrier or excipient. Generally, the formulations are manufactured by homogeneously and thoroughly mixing the active ingredient with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, molding the product.

[0404] The compositions of the present invention can, without limitation, be formulated into any of a number of possible dosage forms, such as tablets, capsules, gel capsules, liquid syrups, softgels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. The aqueous suspension may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0405] C. Additional formulations i. Emulsion The iRNA of the present invention can be prepared as an emulsion and formulated. An emulsion is generally a heterogeneous system in which one liquid is dispersed in another liquid, usually in the form of droplets with a diameter greater than 0.1 μm (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, 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 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). An emulsion is often a two-layer system containing two immiscible liquid phases that are well mixed and dispersed from each other. Generally, emulsions can be either water in oil (w / o) or oil in water (o / w). When the aqueous phase is finely divided and dispersed as microdroplets within the oil phase of the mass, the resulting composition is called a water in oil (w / o) emulsion.Alternatively, when the oil phase is finely divided and dispersed as microdroplets in a large volume of aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion. The emulsion may contain additional components in addition to the dispersed phase and active drug, which may exist as solutions in the aqueous phase, the oil phase, or as separate phases themselves. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions can also be multilayer emulsions consisting of three or more phases, such as oil-in-water (o / w / o) and water-in-oil (w / o / w) emulsions. Such complex formulations often offer certain advantages not provided by simple two-component emulsions. A multilayer emulsion in which individual oil droplets of an o / w emulsion surround smaller water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets surrounded by water droplets stabilized in a continuous phase of oil provides an o / w / o emulsion.

[0406] Emulsions are characterized by having little to no thermodynamic stability. Often, the dispersion or discontinuous phase of an emulsion is well dispersed externally or within a continuous phase and maintained in this form using an emulsifier or the viscosity of the formulation. Any of the emulsion phases may be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Other means of stabilizing an emulsion include the use of emulsifiers that may be incorporated into any of the phases of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorbent bases, and finely dispersed solids (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0407] Synthetic surfactants, also known as surfactants, have found broad applicability in emulsion formulation and are outlined in the literature (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; 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 typically amphiphilic and contain both hydrophilic and hydrophobic parts. The ratio of hydrophilicity to hydrophobicity in a surfactant is called the hydrophilic / lipophilic balance (HLB), and it is a valuable tool for classifying and selecting surfactants during pharmaceutical preparation. Surfactants can be classified into different types based on the properties of their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (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; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0408] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorbent substrates, such as anhydrous lanolin and hydrophilic petrolatum, take in water to form w / o emulsions, but retain their hydrophilic properties, still maintaining their semi-solid consistency. Micronized solids are used as good emulsifiers, particularly in combination with surfactants and in viscous formulations. These include polar inorganic solids (e.g., heavy metal hydroxides), non-swelling clays (e.g., bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal aluminum magnesium silicate), pigments, and non-polar solids (e.g., carbon or glyceryl tristearate).

[0409] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty 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).

[0410] Hydrophilic colloids include naturally occurring rubbers and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These form colloidal solutions that stabilize emulsions by dispersing in water or swelling in water to form a strong interfacial film around droplets of the dispersed phase, and by increasing the viscosity of the outer phase.

[0411] Because emulsions often contain numerous components such as carbohydrates, proteins, sterols, and phosphatides that can readily support microbial growth, these formulations frequently incorporate preservatives. Commonly used preservatives in formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants, in addition to emulsion formulations, also typically prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, as well as antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0412] The application of emulsion formulations via cutaneous, oral, and parenteral routes, as well as their manufacturing methods, are outlined in the literature (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). Emulsion formulations for oral delivery are widely used due to their ease of formulation, absorption, and bioavailability (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 preparations are commonly found in materials administered orally as o / w emulsions.

[0413] ii. Microemulsion In one embodiment of the present invention, iRNA is formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and an amphiphilic substance that is a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, 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 the oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally an alcohol of intermediate chain length, to form a clear system. Therefore, microemulsions are described as thermodynamically stable, isotropic, transparent dispersions consisting 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, pages 185-215). Microemulsions are typically prepared using a combination of 3 to 5 components, including oil, water, surfactant, auxiliary surfactant, and electrolyte.Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used, as well as the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0414] Phenomenological methods using phase diagrams have been extensively studied and provide those skilled in the art with a broad understanding 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. 33). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a thermodynamically stable droplet formulation that forms spontaneously.

[0415] Surfactants used in the preparation of microemulsions include, without limitation, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with auxiliary surfactants. Auxiliary surfactants, typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, play a role in increasing interfacial fluidity by penetrating the surfactant film and consequently forming an irregular film through the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of auxiliary surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically, but not limited to, be water, an aqueous solution of a drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can, but not limited to, include materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolated glycerides, saturated polyglycolated C8-C10 glycerides, vegetable oils, and silicone oils.

[0416] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to improve the oral bioavailability of peptide-containing drugs (see, for example, U.S. Patent Nos. 6,191,105, 7,063,860, 7,070,802, and 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as improved drug solubilization, protection of drugs from enzymatic hydrolysis, potential for enhanced drug absorption due to altered membrane fluidity and permeability induced by surfactants, ease of preparation, ease of oral administration beyond solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-14). In many cases, microemulsions can form spontaneously at ambient temperature when their components are brought together. This can be particularly advantageous when formulating heat-sensitive drugs, peptides, or iRNAs. Microemulsions are also effective for the efficient delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to promote increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improved local cellular uptake of iRNAs and nucleic acids.

[0417] The microemulsion of the present invention may include additional components and additives such as sorbitan monostearate (Grill® 3), Labrasol®, and permeation enhancers to improve the formulation properties and enhance the absorption of iRNA and nucleic acids of the present invention. The permeation enhancers used in the microemulsion of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of these classes is outlined above.

[0418] iii. Particulates The iRNA of the present invention may be incorporated into particles, such as microparticles. Microparticles may be produced by spray drying, but may also be produced by other methods including freeze-drying, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0419] iv. Penetration enhancers In one embodiment, the present invention uses various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily pass through cell membranes. It has been found that non-lipophilic drugs can also pass through cell membranes if the membrane being penetrated is treated with a penetration enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the penetration of lipophilic drugs.

[0420] Penetration enhancers can be classified as be...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910 of Sequence ID No.

1. A double-stranded ribonucleic acid (dsRNA) agent comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from one of the nucleotide sequences 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO:

2.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene, wherein the dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region containing at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in Table 3 or 5 by three or fewer nucleotides.

3. The antisense chains are AD-72506, AD-72319, AD-72502, AD-72513, AD-72499, AD-72303, AD-72500, AD-72522, AD-72512, AD-72304, AD-72514, AD-72257, AD-72295, AD-72332, AD-72507, AD-72311, AD-72501, AD-72508, AD-72293, AD-72322, AD-72264, AD-72290, A The dsRNA agent according to claim 2, comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one double-stranded antisense sequence selected from the group consisting of D-72338, AD-72315, AD-72272, AD-72337, AD-72298, AD-72503, AD-72327, AD-72521, AD-72309, AD-72313, AD-72517, AD-72316, AD-72335, or AD-72317.

4. A dsRNA agent according to any one of claims 1 to 3, wherein the sense and antisense strands include sequences selected from any of the sequences in Table 3 or 5.

5. A dsRNA agent according to any one of claims 1 to 4, wherein the dsRNA comprises at least one modified nucleotide.

6. A dsRNA agent according to any one of claims 1 to 4, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified.

7. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene, wherein the dsRNA agent includes a sense strand and an antisense strand that form a double-stranded region. The sense strand consists of nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, 959-977, 992-1010, 922-1041, 1013- The antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from one of the nucleotide sequences 1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, and the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 2, Substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides. The sense chain is conjugated to a ligand bound to its 3' end. Double-stranded ribonucleic acid (dsRNA) agent.

8. The dsRNA agent according to claim 7, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

9. The dsRNA agent according to claim 7, wherein at least one modified nucleotide is selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino-modified 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 non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic.

10. The dsRNA agent according to claim 9, wherein the modified nucleotide includes a short sequence of a 3' terminal deoxythymine nucleotide (dT).

11. The dsRNA agent according to claim 2 or 3, wherein the complementary region has a length of at least 17 nucleotides.

12. The dsRNA agent according to claim 2 or 3, wherein the complementary region has a nucleotide length of 19 to 21 nucleotides.

13. The dsRNA agent according to claim 12, wherein the complementary region has a nucleotide length of 19.

14. A dsRNA agent according to any one of claims 1 to 3 and 7, wherein each chain has a length of 30 nucleotides or less.

15. A dsRNA agent according to any one of claims 1 to 3 and 7, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

16. A dsRNA agent according to any one of claims 1 to 3 and 7, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

17. A dsRNA agent according to any one of claims 1 to 3, further comprising a ligand.

18. The dsRNA agent according to claim 17, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

19. The dsRNA agent according to claim 7 or 17, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

20. The ligand is 【Chemistry 1】 The dsRNA agent according to claim 19.

21. The dsRNA agent is used in the following scheme: 【Chemistry 2】 (In the formula, X is either O or S) The dsRNA agent according to claim 19, which is conjugated as shown.

22. The dsRNA agent according to claim 21, wherein X is O.

23. The dsRNA agent according to claim 2 or 3, wherein the complementary region includes one of the antisense sequences in Table 3 or 5.

24. The dsRNA agent according to claim 2 or 3, wherein the complementary region comprises one of the antisense sequences in Table 3 or 5.

25. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region to the mRNA encoding KHK, and each strand having a length of about 14 to about 30 nucleotides, and having 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) (wherein i, j, k, and l are independently 0 or 1; p, p', q, and q' are independent of each other and range from 0 to 6; Each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, unmodified, or a combination thereof; n each ｐ , n ｐ ', n ｑ and n ｑ ' may or may not be present, and these independently indicate overhang nucleotides; Each of XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' independently represents one motif of three identical modifications on three consecutive nucleotides; N ｂ Unlike the modification above, the modification above is N ｂ The above modifier differs from the above modifier of Y; and The sense strand is conjugated to at least one ligand. A double-stranded ribonucleic acid (dsRNA) agent, as shown by [the relevant source].

26. The dsRNA agent according to claim 25, 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.

27. The dsRNA agent according to claim 25, 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.

28. The dsRNA agent according to claim 25, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

29. The dsRNA agent according to claim 25, wherein the YYY motif is present at or near the sense strand cleavage site.

30. The dsRNA agent according to claim 25, wherein the Y'Y'Y' motif is located at positions 11, 12, and 13 from the 5' end of the antisense strand.

31. The dsRNA agent according to claim 30, wherein Y' is 2'-O-methyl.

32. Equation (III) is Equation (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 dsRNA agent according to claim 29, as shown by [representation].

33. Equation (III) is given by equation (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 indicates an oligonucleotide sequence containing 1 to 5 modified nucleotides.) The dsRNA agent according to claim 29, as shown by [representation].

34. Equation (III) is given by equation (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 dsRNA agent according to claim 29, as shown by [representation].

35. Equation (III) is, Equation (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, each N ａ and N ａ (' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides.) The dsRNA agent according to claim 29, as shown by [representation].

36. The dsRNA agent according to claim 7 or 25, wherein the double-stranded region has a length of 15 to 30 nucleotide pairs.

37. The dsRNA agent according to claim 36, wherein the double-stranded region has a length of 17 to 23 nucleotide pairs.

38. The dsRNA agent according to claim 36, wherein the double-stranded region has a length of 17 to 25 nucleotide pairs.

39. The dsRNA agent according to claim 36, wherein the double-stranded region has a length of 23 to 27 nucleotide pairs.

40. The dsRNA agent according to claim 36, wherein the double-stranded region has a length of 19 to 21 nucleotide pairs.

41. The dsRNA agent according to claim 7 or 25, wherein the double-stranded region has a length of 21 to 23 nucleotide pairs.

42. The dsRNA agent according to claim 25, wherein each chain has a nucleotide length of 15 to 30.

43. A dsRNA agent according to any one of claims 7, 25, and 35, wherein each chain has a nucleotide length of 19 to 30 nucleotides.

44. The dsRNA agent according to claim 7 or 25, wherein the nucleotide modification is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

45. The dsRNA agent according to claim 44, wherein the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

46. The dsRNA agent according to claim 7 or 25, wherein the ligand is one or more GalNAc derivatives linked via monovalent, divalent, or trivalent branched linkers.

47. The ligand is 【Transformation 3】 The dsRNA agent according to claim 25.

48. The dsRNA agent according to claim 25, wherein the ligand is bound to the 3' end of the sense strand.

49. RNAi agents are used in the following scheme: 【Chemistry 4】 A dsRNA agent according to claim 48, which is conjugated to a ligand shown in [reference].

50. The dsRNA agent according to claim 7 or 25, wherein the agent further comprises a bond between at least one phosphorothioate or methylphosphonate nucleotide.

51. The dsRNA agent according to claim 50, wherein the bond between the phosphorothioate or methylphosphonate nucleotides is located at the 3' end of a single strand.

52. The dsRNA agent according to claim 51, wherein the chain is an antisense chain.

53. The dsRNA agent according to claim 51, wherein the strand is a sense strand.

54. The dsRNA agent according to claim 50, wherein the bond between the phosphorothioate or methylphosphonate nucleotides is located at the 5' end of a single strand.

55. The dsRNA agent according to claim 54, wherein the chain is an antisense chain.

56. The dsRNA agent according to claim 54, wherein the strand is a sense strand.

57. The dsRNA agent according to claim 50, wherein the bond between the phosphorothioate or methylphosphonate nucleotide is present at both the 5' and 3' ends of the single strand.

58. The dsRNA agent according to claim 57, wherein the chain is an antisense chain.

59. The dsRNA agent according to claim 7 or 25, wherein one base pair at the 5' end of the double-stranded antisense strand is an AU base pair.

60. The dsRNA agent according to claim 25, wherein the Y nucleotide includes a 2'-fluoro modification.

61. The dsRNA agent according to claim 25, wherein the Y' nucleotide includes a 2'-O-methyl modification.

62. The dsRNA agent according to claim 25, wherein p' > 0.

63. The dsRNA agent according to claim 25, wherein p' = 2.

64. The dsRNA agent according to claim 63, wherein q' = 0, p = 0, q = 0, and the p' overhang nucleotide is complementary to the target mRNA.

65. The dsRNA agent according to claim 63, wherein q' = 0, p = 0, q = 0, and the p' overhang nucleotide is non-complementary to the target mRNA.

66. The dsRNA agent according to claim 57, wherein the sense strand has all 21 nucleotides and the antisense strand has all 23 nucleotides.

67. at least one n ｐ The dsRNA agent according to any one of claims 62 to 66, wherein the ' is bound to an adjacent nucleotide via a phosphorothioate bond.

68. All n ｐ The dsRNA agent according to claim 67, wherein the ' is bound to an adjacent nucleotide via a phosphorothioate bond.

69. The dsRNA agent according to claim 25, wherein the RNAi agent is selected from the group of RNAi agents listed in Table 3 or 5.

70. The dsRNA agent according to claim 25, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

71. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand, and the sense strand comprises nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 8 The dsRNA agent comprises at least 14 consecutive nucleotides of one of the following nucleotide sequences: 92-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand contains a complementary region to the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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 independently 0 or 1; p, p', q, and q' are independent of each other and range from 0 to 6; Each N ａ and N ａ ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, unmodified, or a combination thereof; Each n ｐ 、n ｐ ', n ｑ and n ｑ ' may be present or absent, and these independently represent overhang nucleotides; Each of XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' independently represents one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ Unlike the modification above, the modification above is N ｂ The above modifier differs from the above modifier of Y; and The sense strand is conjugated to at least one ligand. A dsRNA agent as shown by [the specified method].

72. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855 of Sequence ID No.

1. The dsRNA agent comprises at least 14 consecutive nucleotides of any one nucleotide sequence of 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand contains a complementary region to the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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 independently 0 or 1; n each ｐ , n ｑ and n ｑ ' may or may not be present, and these independently indicate an overhanging nucleotide; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is attached to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, 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, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ Unlike the modification above, the modification above is N ｂ The above modifier differs from the above modifier of Y; and The sense strand is conjugated to at least one ligand. A double-stranded ribonucleic acid (dsRNA) agent, as shown by [the relevant source].

73. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855 of Sequence ID No.

1. The dsRNA agent comprises at least 14 consecutive nucleotides of any one nucleotide sequence of 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand contains a complementary region to the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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 either 0 or 1; each n ｐ , n ｑ and n ｑ ' may or may not be present, and these independently indicate overhang nucleotides; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is attached to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, 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, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ Unlike the modification above, the modification above is N ｂ The above modifier differs from the above modifier of Y; and The sense chain is conjugated to at least one ligand, where the ligand is one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. A double-stranded ribonucleic acid (dsRNA) agent, as shown by [the relevant source].

74. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855 of Sequence ID No.

1. The dsRNA agent comprises at least 14 consecutive nucleotides of any one nucleotide sequence of 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand contains a complementary region to the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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 either 0 or 1; n each ｐ , n ｑ and n ｑ ' may or may not be present, and these independently indicate overhang nucleotides; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is attached to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified, 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, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ Unlike the modification above, the modification above is N ｂ The above modifier differs from the above modifier; The sense chain comprises at least one phosphorothioate linkage, the sense chain is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a monovalent, divalent, or trivalent branched linker. A double-stranded ribonucleic acid (dsRNA) agent, as shown by [the relevant source].

75. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene in cells, wherein the dsRNA agent comprises a sense strand and an antisense strand, the sense strand comprising nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855 of Sequence ID No.

1. The dsRNA agent comprises at least 14 consecutive nucleotides of any one nucleotide sequence of 892-910, 959-977, 992-1010, 922-1041, 1013-1041, 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294, or 1320-1350, wherein the antisense strand contains a complementary region to the mRNA encoding KHK, and each strand is approximately 14 to approximately 30 nucleotides long, where the dsRNA agent is given 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, n each ｐ , n ｑ and n ｑ ' may or may not be present, and independently indicates an overhang nucleotide; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is attached to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents oligonucleotide sequences containing 0 to 25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; The sense chain contains at least one phosphorothioate bond; The sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via monovalent, divalent, or trivalent branched linkers. A double-stranded ribonucleic acid (dsRNA) agent, as shown by [the relevant source].

76. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of the ketohexokinase (KHK) gene in cells, wherein the dsRNA agent includes a sense strand and an antisense strand that form a double-stranded region. The sense strand consists of nucleotides 89-107, 176-194, 264-282, 474-492, 508-526, 529-547, 562-580, 616-646, 682-700, 705-723, 705-757, 705-799, 739-757, 739-799, 760-799, 804-822, 837-855, 892-910, 959-977, 992-1010, 922-1041, 1013-10 41, comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one nucleotide sequence of 1069-1108, 1169-1140, 1111-1140, 1155-1196, 1221-1261, 1267-1294 or 1320-1350, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2, Substantially all nucleotides of the sense strand include modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications; The sense strand contains two phosphorothioate nucleotide interlinks at its 5' end; Substantially all nucleotides of the antisense strand include modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications; The antisense strand contains two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end; and The sense chain is conjugated to one or more GalNAc derivatives linked at its 3' end via a monovalent, divalent, or trivalent branched linker. A double-stranded ribonucleic acid (dsRNA) agent, as shown in [reference].

77. The dsRNA agent according to claim 76, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

78. The dsRNA agent according to claim 76, wherein each chain has a nucleotide length of 19 to 30 nucleotides.

79. Cells comprising a dsRNA agent according to any one of claims 1 to 3, 7, 25, or 70 to 75.

80. A pharmaceutical composition for inhibiting the expression of a ketohexokinase (KHK) gene, comprising a dsRNA agent according to any one of claims 1 to 3, 7, 24, or 70 to 75.

81. A pharmaceutical composition comprising a dsRNA agent and a lipid preparation according to any one of claims 1 to 3.

82. The pharmaceutical composition according to claim 81, wherein the lipid preparation comprises LNP or MC3.

83. A method for inhibiting the expression of the ketohexokinase (KHK) gene in cells, (a) a step of contacting cells with a double-stranded RNAi agent according to any one of claims 1 to 3, 7, 25 and 71 to 76 or a pharmaceutical composition according to any one of claims 80 to 82; and (b) A step of maintaining the cells generated in step (a) for a sufficient time to obtain degradation products of the mRNA transcript of the KHK gene, thereby inhibiting the expression of the KHK gene in the cells. Methods that include...

84. The method according to claim 83, wherein the cells are located within the target.

85. The method according to claim 84, wherein the subject is a human.

86. The method according to any one of claims 83 to 85, wherein KHK expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or below the detection limit compared to a suitable control.

87. A method for treating a subject having a disease or disorder that benefits from reduced expression of ketohexokinase (KHK), characterized by administering a therapeutically effective amount of a double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 3, 7, 25 and 71 to 76 or a pharmaceutical composition according to any one of claims 80 to 82 to the subject, thereby treating the subject.

88. A method for preventing at least one symptom in a subject having a disease or disorder that benefits from reduced expression of ketohexokinase (KHK), characterized by administering a preventively effective amount of a dsRNA agent according to any one of claims 1 to 3, 7, 25 and 71 to 76 or a pharmaceutical composition according to any one of claims 80 to 82 to the subject, thereby preventing at least one symptom in the subject having a disorder that benefits from reduced expression of KHK.

89. The method according to claim 87 or 88, wherein a decrease in fructose metabolism occurs when dsRNA is administered as the target.

90. The method according to claim 87 or 88, wherein the disorder is a KHK-related disease.

91. The method according to claim 90, wherein the KHK-related disease includes hyperuricemia.

92. The method according to claim 90, wherein the KHK-related disease is gout.

93. The method according to claim 90, wherein the KHK-related disease includes liver disease.

94. The method according to claim 93, wherein the liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

95. The method according to claim 90, wherein the KHK-related disease includes dyslipidemia or abnormal lipid deposition or dysfunction.

96. The method according to claim 95, wherein the dyslipidemia includes one or more hyperlipidemia, high LDL cholesterol, low HDL cholesterol, high triglycerides, postprandial hypertriglycerides, adipocyte dysfunction, visceral fat deposition, obesity, and metabolic syndrome.

97. The method according to claim 90, wherein the KHK-related disease includes impaired blood glucose control.

98. The method according to claim 97, wherein the impaired blood glucose control includes one or more insulin resistances, type II diabetes, and glucose intolerance that are not related to an immune response to insulin.

99. The method according to claim 90, wherein the KHK-related disease includes kidney disease.

100. The method according to claim 93, wherein the liver disease comprises at least one acute kidney injury, tubular dysfunction, inflammatory changes in the proximal tubules, and chronic kidney disease.

101. The method according to claim 90, wherein the KHK-related disease includes cardiovascular disease.

102. The method according to claim 101, wherein the cardiovascular disease comprises at least one hypertension and endothelial cell dysfunction.

103. The method according to any one of claims 87 to 102, wherein the subject is a human.

104. The method according to claim 103, wherein the subject has or is prone to renal dysfunction.

105. The method according to any one of claims 87 to 104, further comprising administering an agent for treating KHK-related diseases.

106. The method according to any one of claims 87 to 105, wherein the dsRNA agent is administered in a dose of approximately 0.01 mg / kg to approximately 50 mg / kg.

107. The method according to any one of claims 87 to 106, wherein the dsRNA agent is administered subcutaneously to the subject.

108. The method according to any one of claims 87 to 107, further comprising measuring the level of KHK in the subject.

109. The method according to any one of claims 87 to 108, further comprising measuring the level of fructose metabolism in the subject.

110. The method according to any one of claims 87 to 109, further comprising measuring the uric acid level in the subject.

111. The method according to any one of claims 87 to 110, further comprising measuring serum lipid levels in a subject.

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