Angiopoietin-Like 3 (ANGPTL3) iRNA Compositions and Methods of Use Thereof

Abstract

The present invention provides iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the ANGPL3 gene. [Solution] A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of ANGPTL3, the dsRNA comprising 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 no more than 3 nucleotides, and the antisense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 5 by no more than 3 nucleotides.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 61 / 499,620, filed June 21, 2011, and U.S. Provisional Patent Application No. 61 / 638,288, filed April 25, 2012, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted factors that regulate lipid metabolism and are expressed primarily in the liver (Non-Patent Document 1). ANGPTL3 dually inhibits the catalytic activity of lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and endothelial lipase (EL), which hydrolyzes high-density lipoprotein (HDL) phospholipids. In KK / Snk mice that are still obese but have reduced lipids, reduced ANGPTL3 expression has a protective effect against hyperlipidemia and atherosclerosis by promoting the clearance of triglycerides (Non-Patent Document 2). Human ANGPTL3 plasma concentrations are positively correlated with plasma HDL cholesterol and HDL phospholipid levels (Non-Patent Document 3).

[0003] Lipid metabolism disorders can increase the levels of serum lipids, such as triglycerides and / or cholesterol. Elevated serum lipids are strongly associated with hypertension, cardiovascular disease, diabetes and other conditions. Hypertriglyceridemia is an example of a lipid metabolism disorder characterized by high blood levels of triglycerides. Hypertriglyceridemia has been associated with atherosclerosis, even in the absence of high cholesterol levels (hypercholesterolemia). When triglyceride levels are excessive (i.e., greater than 1000 mg / dl or 12 mmol / l), hypertriglyceridemia can also cause pancreatitis. Hyperlipidemia is another example of a lipid metabolism disorder characterized by elevated levels of any or all of lipids and / or lipoproteins in the blood. Current treatments for lipid metabolism disorders, including diet, exercise, and treatment with statins and other drugs, are not always effective. Thus, there is a need in the art for alternative treatments for subjects suffering from lipid metabolism disorders. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Koishi, R. et al., (2002) Nat. Genet. 30(2):151-157 [Non-Patent Document 2] Ando et al.,(2003)J.Lipid Res.,44:1216-1223 [Non-Patent Document 3] Shimamura et al.,(2007)Arterioscler.Thromb.Vasc.Biol.,27:366-372 Summary of the Invention [Means for solving the problem]

[0005] The present invention provides an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of ANGPL3 gene. The ANGPL3 gene can be in a cell, for example, in a subject, such as a human. The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of ANGPL3 gene and / or to treat a subject who may benefit from inhibiting or reducing the expression of ANGPL3 gene, for example, a subject who suffers from or is susceptible to a lipid metabolism disorder, such as a subject who suffers from or is susceptible to hyperlipidemia or hypertriglyceridemia.

[0006] Thus, in one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of ANGPTL3. The dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:5 by no more than 3 nucleotides.

[0007] In another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of ANGPTL3. The dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a region of complementarity comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10 by no more than 3 nucleotides.

[0008] In one embodiment, the sense and antisense strands comprise a sequence selected from the group consisting of AD-53063.1, AD-53001.1, AD-53015.1, AD-52986.1, AD-52981.1, AD-52953.1, AD-53024.1, AD-53033.1, AD-53030.1, AD-53080.1, AD-53073.1, AD-53132.1, AD-52983.1, AD-52954.1, AD-52961.1, AD-52994.1, AD-52970.1, AD-53075.1, AD-53147.1, AD-53077.1 in Tables 7 and 8.

[0009] In certain embodiments of the present invention, the dsRNA comprises at least one modified nucleotide. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of 2'-O-methyl modified nucleotides, nucleotides comprising 5'-phosphorothioate groups, and terminal nucleotides linked to cholesteryl derivatives or dodecanoic acid bisdecylamide groups. In another embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides comprising unnatural bases.

[0010] The region of complementarity of the dsRNA can be at least 17 nucleotides in length, 19-21 nucleotides in length, or 19 nucleotides in length.

[0011] In one embodiment, each strand of the dsRNA is 30 nucleotides or less in length.

[0012] At least one strand of the dsRNA can include a 3' overhang of at least one nucleotide or at least two nucleotides.

[0013] In certain embodiments, the dsRNA further comprises a ligand. In one embodiment, the ligand is attached to the 3' end of the sense strand of the dsRNA.

[0014] In certain embodiments, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached via a bivalent or trivalent branched linker. [ka] It is.

[0015] In one embodiment, the RNAi agent was conjugated to a ligand as shown in the schematic diagram below. [ka]

[0016] In some embodiments, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. In some embodiments, the strand is the antisense strand. In other embodiments, the strand is the sense strand.

[0017] In one embodiment, the region of complementarity of the dsRNA consists of one of the antisense sequences in Tables 2, 3, 7, 8, 9 and 10.

[0018] In another embodiment, the dsRNA comprises a sense strand consisting of a sense strand sequence selected from the sequences of Tables 2, 3, 7, 8, 9, and 10, and an antisense strand consisting of an antisense sequence selected from the sequences of Tables 2, 3, 7, 8, 9, and 10.

[0019] In another embodiment, the invention provides a cell, eg, a liver cell, comprising a dsRNA of the invention.

[0020] In yet another aspect, the invention provides a vector encoding at least one strand of a dsRNA, where the dsRNA comprises a region of complementarity to at least a portion of an mRNA encoding ANGPTL3, the dsRNA is 30 base pairs or less in length, and the dsRNA targets the mRNA for cleavage. The region of complementarity can be at least 15 nucleotides in length, or 19-21 nucleotides in length.

[0021] In a further aspect, the invention provides a cell comprising a vector encoding at least one strand of a dsRNA, where the dsRNA comprises a region of complementarity to at least a portion of an mRNA encoding ANGPTL3, the dsRNA is 30 base pairs or less in length, and the dsRNA targets the mRNA for cleavage.

[0022] In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of the ANGPTL3 gene, the pharmaceutical composition comprising a dsRNA or vector of the present invention.

[0023] In one embodiment, the pharmaceutical composition comprises a lipid formulation, such as an MC3, SNALP or XTC formulation.

[0024] In another aspect, the present invention provides a method for inhibiting the expression of ANGPTL3 in a cell, the method comprising contacting the cell with a dsRNA or vector of the present invention, and maintaining the resulting cell for a sufficient time to obtain degradation of the mRNA transcript of ANGPTL3 gene, thereby inhibiting the expression of ANGPTL3 gene in the cell.

[0025] The cell can be in a subject, such as a human subject, for example, a human subject suffering from a lipid metabolism disorder, for example hyperlipidemia or hypertriglyceridemia.

[0026] In one embodiment of the methods of the invention, expression of ANGPTL3 is inhibited by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0027] In another aspect, the present invention provides a method for treating a subject suffering from a disorder that can benefit from the reduction of ANGPTL3 expression, for example, a lipid metabolism disorder, such as hyperlipidemia or hypertriglyceridemia.The method comprises administering a therapeutically effective amount of the dsRNA or vector of the present invention to the subject, thereby treating the subject.

[0028] The disorder can be a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.

[0029] In one embodiment, administering dsRNA to a subject causes the level of serum lipids, triglycerides, cholesterol and / or free fatty acids to be reduced; and / or the accumulation of ANGPTL3 protein to be reduced.In one embodiment, administering dsRNA to a subject causes the level of LDL-C, HDL-C, VLDL-C, IDL-C and / or total cholesterol to be reduced.

[0030] In one embodiment, the dsRNA is administered at a concentration of about 0.01 mg / kg to about 10 mg / kg, for example, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 10 mg / kg, or about 0.3 mg / kg to about 5 mg / kg. g, about 0.3 mg / kg to about 10 mg / kg, about 0.4 mg / kg to about 5 mg / kg, about 0.4 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg ~10mg / kg, 1mg / kg~5mg / kg, 1mg / kg~10mg / kg, 1.5mg / kg~5mg / kg, 1.5mg / kg~10mg / kg, 2mg / k g ~ approx. 2.5 mg / kg, approx. 2 mg / kg ~ approx. 10 mg / kg, approx. 3 mg / kg ~ approx. 5 mg / kg, approx. 3 mg / kg ~ approx. 10 mg / kg, approx. 3.5 mg / kg ~ approx. 5 mg / kg, approx. 4 mg / kg~about 5mg / kg, about 4.5mg / kg~about 5mg / kg, about 4mg / kg~about 10mg / kg, about 4.5mg / kg~about 10mg / kg, about 5mg / kg~about 10mg / kg, about 5. The dose is 5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0031] For example, the dsRNA may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8.4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8.8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8.9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0032] In another embodiment, the dsRNA is about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 ... 0mg / kg, about 30 to about 50mg / kg, about 35 to about 50mg / kg, about 40 to about 50mg / kg, about 45 to about 50mg / kg, about 0.5 to about 45mg / kg, about 0.75 to about 45mg / kg, about 1 to about 45mg / mg, about 1.5 to about 45mg / kb, about 2 to about 45mg / k g, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg mg, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 ~40mg / kg, 15~40mg / kg, 20~40mg / kg, 25~40mg / kg, 25~40mg / kg, 30~40mg / kg, 35~40mg / kg, 0.5~30mg / kg, 0.75~30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.It is administered at a dose of about 5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Intermediate values ​​and ranges of the values ​​described are also intended to be part of the present invention. .

[0033] For example, subjects may have approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3. 9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8.4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 .7, 7.8.7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8.8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8.9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, Treatment amounts of iRNA such as 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg may be administered. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0034] In another aspect, the present invention provides a method for inhibiting the expression of ANGPTL3 in a subject. The method comprises administering a therapeutically effective amount of a dsRNA or vector of the present invention to a subject, thereby inhibiting the expression of ANGPTL3 in the subject.

[0035] In yet another embodiment, the present invention provides a kit for carrying out the method of the present invention.In one embodiment, the present invention provides a kit for carrying out the method of inhibiting the expression of ANGPTL3 gene in a cell by contacting the cell with a double-stranded RNAi agent in an amount effective to inhibit the expression of ANGPTL3 in the cell.The kit includes an RNAi agent and instructions for use, and optionally, a means for administering the RNAi agent to a subject. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 is a schematic diagram of the experimental procedure used in the in vivo studies described in Example 2. [Diagram 2] Panel A of Figure 2 is a graph showing measured levels of ANGPTL3 protein in wild-type (WT) mice after treatment with the indicated iRNAs or controls. Panel B of Figure 2 is a graph showing measured levels of ANGPTL3 protein in ob / ob mice after treatment with the indicated iRNAs or controls. [Diagram 3] Panel A of Figure 3 is a graph showing measured levels of LDL-c in wild-type (WT) mice following treatment with the indicated iRNA or a control. Panel B of Figure 3 is a graph showing measured levels of LDL-c in ob / ob mice following treatment with the indicated iRNA or a control. [Figure 4] Figure 4, Panel A, is a graph showing the measured levels of triglycerides in wild-type (WT) mice following treatment with the indicated iRNAs or controls. Figure 4, Panel B, is a graph showing the measured levels of triglycerides in ob / ob mice following treatment with the indicated iRNAs or controls. [Diagram 5]Panel A of Figure 5 is a graph showing measured levels of total cholesterol (TC) in wild type (WT) mice following treatment with the indicated iRNAs or controls. Panel B of Figure 5 is a graph showing measured levels of total cholesterol (TC) in ob / ob mice following treatment with the indicated iRNAs or controls. [Figure 6] Panel A of Figure 6 is a graph showing measured levels of HDL-c in wild-type (WT) mice following treatment with the indicated iRNA or a control. Panel B of Figure 6 is a graph showing measured levels of HDL-c in ob / ob mice following treatment with the indicated iRNA or a control. [Figure 7] Graph showing measured levels of ANGPTL3 protein in human PCS transgenic mice following treatment with a single dose of the indicated iRNA or control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention provides an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of ANGPTL3 gene.The ANGPTL3 gene can be in a cell, for example, in a subject, such as a human.The present invention also provides a method of using the iRNA composition of the present invention to inhibit the expression of ANGPTL3 gene and / or to treat a subject suffering from a disorder that can benefit from inhibiting or reducing the expression of ANGPTL3 gene, for example, a lipid metabolism disorder, such as hyperlipidemia or hypertriglyceridemia.

[0038] The iRNA of the present invention is about 30 nucleotides in length or less, for example, 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 2 The iRNAs include an RNA strand (antisense strand) having a region that is 4, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of the mRNA transcript of the ANGPTL3 gene. The use of these iRNAs allows for targeted degradation of the mRNA of the ANGPTL3 gene in mammals. Very low doses of ANGPTL3 iRNA, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of the expression of the ANGPTL3 gene. Using cell-based assays, the present inventors have demonstrated that iRNA targeting ANGPTL3 can mediate RNAi, resulting in significant inhibition of the expression of ANGPTL3 gene.Therefore, the methods and compositions comprising these iRNAs are useful for treating subjects who can benefit from the reduction of the level and / or activity of ANGPTL3 protein, such as subjects suffering from lipid metabolism disorders, such as hyperlipidemia or hypertriglyceridemia.

[0039] The following detailed description discloses methods for making and using compositions containing iRNA to inhibit expression of the ANGPTL3 gene, as well as compositions and methods for treating subjects suffering from diseases and disorders that may benefit from inhibition and / or reduction of expression of this gene.

[0040] I. Definition So that the present invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a variable is described, it is intended that values ​​and ranges intermediate to the described values ​​are also part of the present invention.

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0042] The term "including" is used herein to mean, and is used synonymously with, the phrase "including but not limited to." The term "or" is used herein to mean, and is used synonymously with, the term "and / or," unless the context clearly indicates otherwise.

[0043] The term "ANGPTL3" refers to angiopoietin-like protein 3, having an amino acid sequence derived from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, pig, sheep, primate, monkey, and guinea pig, unless otherwise specified. The term also refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of native ANGPTL3. The term encompasses the full-length unprocessed precursor form of ANGPTL3 as well as the mature form resulting from post-translational cleavage of the signal peptide and the form resulting from proteolytic processing of the fibrinogen-like domain. The sequence of human ANGPTL3 mRNA transcript can be found, for example, in GenBank Accession No. GI:41327750 (NM_014495.2; SEQ ID NO:1). The predicted sequence of rhesus monkey ANGPTL3 mRNA can be found, for example, in GenBank Accession No. GI:297278846 (XM_001086114.2; SEQ ID NO:2). The sequence of mouse ANGPTL3 mRNA can be found, for example, in GenBank Accession No. GI:142388354 (NM_013913.3; SEQ ID NO:3). The sequence of rat ANGPTL3 mRNA can be found, for example, in GenBank Accession No. GI:68163568 (NM_001025065.1; SEQ ID NO:4).

[0044] The term "ANGPTL3" as used herein also refers to a specific polypeptide expressed in cells due to the mutation of the natural DNA sequence of ANGPTL3 gene, such as single nucleotide polymorphism in ANGPTL3 gene.Many SNPs in ANGPTL3 gene have been identified, and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).Non-limiting examples of SNPs in ANGPTL3 gene can be found in NCBI dbSNP accession numbers rs193064039;rs192778191;rs192764027;rs192528948;rs191931953;rs191293319;rs191171206;rs191145608;rs191086880;rs191012841;or rs190255403.

[0045] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of ANGPTL3 gene, including the mRNA that is the product of RNA processing of the primary transcript.In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near the corresponding portion of the nucleotide sequence of an mRNA molecule formed during transcription of ANGPTL3 gene.

[0046] The target sequence can be about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 1 The length may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Ranges and lengths intermediate to the above listed ranges and lengths are also contemplated as part of the invention.

[0047] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by a sequence shown using standard nucleotide nomenclature.

[0048] "G", "C", "A", "T" and "U" each generally represent a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide or a surrogate replacement moiety, as described in more detail below. Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced by other moieties without significantly changing the base pairing properties of an oligonucleotide that contains a nucleotide with such a replacement moiety. For example, but not limited to, a nucleotide that contains inosine as its base can base pair with a nucleotide that contains adenine, cytosine or uracil. Thus, a nucleotide that contains uracil, guanine or adenine can be replaced by a nucleotide that contains inosine, for example, in the nucleotide sequence of a dsRNA featured in the present invention. In another example, adenines and cytosines anywhere in the oligonucleotide may be substituted with guanines and uracils, respectively, to form GU wobble base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in the present invention.

[0049] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent", as used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as the term is defined herein. iRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of ANGPTL3 in a cell, e.g., a cell in a subject, such as a mammalian subject.

[0050] In one embodiment, the RNAi agent of the present invention comprises a single stranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL3 target mRNA sequence, leading to cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 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). Then, siRNA is incorporated into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex to allow complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to the appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Here, in one aspect, the present invention relates to single-stranded RNA (siRNA) that is produced in cells and promotes the formation of RISC complex that leads to the silencing of target gene, i.e., ANGPTL3 gene. Therefore, the term "siRNA" is also used herein to refer to the above RNAi.

[0051] In another embodiment, the RNAi agent is a single-stranded antisense RNA molecule. The antisense RNA molecule is complementary to a sequence in the target mRNA. The antisense RNA can stoichiometrically inhibit translation by base pairing to the mRNA and physically interfering with the translation machinery (see Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355). The single-stranded antisense RNA molecule can be about 13 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule can include a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from one of the antisense sequences in Tables 2, 3, 7, 8, 9, and 10.

[0052] In another embodiment, the "iRNA" for use in the compositions and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent", or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which are shown to have a "sense" and an "antisense" orientation to a target RNA, i.e., the ANGPTL3 gene. In one embodiment of the present invention, the double-stranded RNA (dsRNA) causes the degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0053] The double-stranded region may be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and may range from about 9-36 base pairs in length, e.g., about 15-30 base pairs in length, e.g., about 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, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26 The nucleic acid sequence may be about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated as part of the invention.

[0054] The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are thus joined by a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand forming the double-stranded structure, the joined RNA strands are called "hairpin loops". A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.

[0055] When the two substantially complementary strands of dsRNA are comprised by separate RNA molecules, the molecules may be covalently linked, but may not be covalently linked. When the two strands are covalently linked by means other than a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the other strand that forms a double-stranded structure, the linking structure is called a "linker". The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest strand of dsRNA minus the overhang present in the double strand. In addition to the double-stranded structure, RNAi may include one or more nucleotide overhangs.

[0056] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. A nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. An overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhang nucleotide may be present at the 5' end, the 3' end, or both ends of either the antisense strand or the sense strand of a dsRNA.

[0057] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' and / or 5' end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' and / or 5' end. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0058] The term "blunt" or "blunt-ended" as used herein in relation to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of the dsRNA may be blunt. If both ends of the dsRNA are blunt, the dsRNA is said to be blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0059] The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., a dsRNA, that comprises a region that is substantially complementary to a target sequence, e.g., an ANGPTL3 mRNA. As used herein, the term "region of complementarity" refers to a region of an antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an ANGPTL3 nucleotide sequence, as defined herein. When a region of complementarity is not completely complementary to a target sequence, there may be mismatches in the internal or terminal regions of the molecule. In general, the most tolerated mismatches are in the terminal regions, e.g., 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of an iRNA.

[0060] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0061] The term "complementary" as used herein, unless otherwise indicated, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize and form a double-stranded structure with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under a given condition, as understood by those skilled in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered internally, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotides.

[0062] A complementary sequence in an iRNA, for example, in a dsRNA described herein, includes base pairing over the entire length of one or both nucleotide sequences of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, no more than 4, no more than 3, or no more than 2 mismatched base pairs upon hybridization to a duplex of up to 30 base pairs while retaining the ability to hybridize under optimal conditions for their ultimate application, e.g., inhibition of gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered as mismatches for purposes of determining complementarity. For example, for purposes described herein, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length may be referred to as "fully complementary" even if the longer oligonucleotide comprises a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide.

[0063] As used herein, "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairs.

[0064] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to matching bases between the sense and antisense strands of a dsRNA or between the antisense strand of an iRNA and a target sequence, as understood from the context of their use.

[0065] 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 continuous portion of an mRNA of interest (e.g., an mRNA encoding ANGPTL3). For example, a polynucleotide is complementary to at least a portion of an ANGPTL3 mRNA if its sequence is substantially complementary to a continuous portion of an mRNA encoding ANGPTL3.

[0066] Generally, the majority of the nucleotides in each strand are ribonucleotides, however, as described in detail herein, each or both strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. Additionally, an "iRNA" may include ribonucleotides having chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any such modifications when used in an iRNA molecule are encompassed by "iRNA" for purposes of this specification and claims.

[0067] As used herein, the term "inhibit" is used synonymously with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0068] As used herein, the phrase "inhibiting expression of ANGPTL3" includes inhibition of expression of any ANGPTL3 gene (e.g., mouse ANGPTL3 gene, rat ANGPTL3 gene, monkey ANGPTL3 gene, or human ANGPTL3 gene, etc.) as well as variants or mutants of the ANGPTL3 gene that encode the ANGPTL3 protein.

[0069] "Inhibiting expression of the ANGPTL3 gene" includes any level of inhibition of the ANGPTL3 gene, for example, at least partial suppression of expression of the ANGPTL3 gene, such as at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition.

[0070] The expression of ANGPTL3 gene can be evaluated based on the level of any variable related to the expression of ANGPTL3 gene, for example, ANGPTL3 mRNA level or ANGPTL3 protein level. The expression of ANGPTL3 can also be indirectly evaluated based on serum lipids, triglycerides, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids. Inhibition can be evaluated by the absolute or relative level reduction of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, for example, pre-dose baseline level, or the level measured from a similar subject, cell, or sample that is untreated or treated with a control (such as, for example, a buffer only control or an inactive agent control).

[0071] In one embodiment, at least partial suppression of expression of the ANGPTL3 gene is assessed by a reduction in the amount of ANGPTL3 mRNA that can be isolated from or detected in a first cell or cell population in which the ANGPTL3 gene is transcribed and that has been treated to inhibit expression of the ANGPTL3 gene, compared to a second cell or cell population (control cells) that is substantially identical to the first cell or cell population but has not been so treated. The degree of inhibition can be expressed by the following formula:

number

[0072] As used herein, the phrase "contacting a cell with an RNAi agent" such as dsRNA includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo. Contacting can be performed directly or indirectly. Thus, for example, an RNAi agent can be physically contacted with a cell by performing a method individually, or the RNAi agent can be placed in a situation that allows it to contact a cell later or that allows it to contact a cell later.

[0073] The step of contacting the cells in vitro can be performed, for example, by incubating the cells with an RNAi agent. The step of contacting the cells in vivo can be performed, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another site, for example, into the bloodstream or subcutaneous space, so that the RNAi agent will later reach the tissue where the contacted cells are located. For example, the RNAi agent can include and / or be bound to a ligand, for example, GalNAc3, that directs the RNAi agent to the site of interest, for example, the liver. A combination of in vitro and in vivo contact methods is also possible. For example, the cells can also be contacted with the RNAi agent in vitro and then transplanted into a subject.

[0074] In one embodiment, contacting a cell with an iRNA includes "introducing" or "delivering an iRNA into a cell" by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of the iRNA can occur by unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by β-glucan delivery systems such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Patent Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further techniques are described herein below and / or are known in the art.

[0075] The term "SNALP" refers to a stable nucleic acid-lipid particle. SNALPs are lipid vesicles with a small aqueous interior coating that contains a nucleic acid, such as an iRNA or a plasmid into which the iRNA is transcribed. SNALPs are described, for example, in U.S. Patent Publication Nos. 20060240093, 20070135372, and International Application WO2009082817, the entire contents of which are incorporated herein by reference. Examples of "SNALP" formulations are described below.

[0076] As used herein, a "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (such as a duck or a goose). In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that may benefit from a decrease in the expression of ANGPTL3 as described herein; a human being at risk for a disease, disorder, or condition that may benefit from a decrease in the expression of ANGPTL3; a human suffering from a disease, disorder, or condition that may benefit from a decrease in the expression of ANGPTL3; and / or a human being being treated for a disease, disorder, or condition that may benefit from a decrease in the expression of ANGPTL3. As used herein, the term "treat" or "treatment" refers to a beneficial or desired result, including, for example, reducing the level of triglycerides in a subject. The term "treat" or "treatment" also includes, but is not limited to, alleviating or ameliorating one or more symptoms of a lipid metabolism disorder, such as, for example, reducing the size of eruptive xanthomas. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0077] "Reduce" in the context of disease markers or symptoms means a statistically significant reduction in such levels. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to a level that is recognized as being within the normal range of individuals without such disorders. As used herein, "prevention" or "preventing", when used in connection with a disease, disorder, or condition that can benefit from a reduction in the expression of the ANGPTL3 gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, such as high triglyceride levels or eruptive xanthomas. The likelihood of developing high triglyceride levels or eruptive xanthomas is reduced, for example, when an individual with one or more risk factors for high triglyceride levels or eruptive xanthomas does not develop high triglyceride levels or eruptive xanthomas, or develops milder high triglyceride levels or eruptive xanthomas compared to a population with the same risk factors and not receiving the treatment described herein. Non-development of a disease, disorder, or condition, or a reduction in the occurrence of symptoms associated with such disease, disorder, or condition (e.g., at least about 10% by a clinically recognized measure of the disease or disorder), or a delayed onset of symptoms (e.g., by only days, weeks, months, or years) is considered effective prevention.

[0078] As used herein, the term "serum lipids" refers to any major lipid present in blood. Serum lipids may be present in blood in free form or as part of protein complexes, such as lipoprotein complexes. Non-limiting examples of serum lipids include triglycerides and cholesterol, such as total cholesterol (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very-low-density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C).

[0079] As used herein, "lipid metabolism disorder" refers to any disorder related to or caused by lipid metabolism disorder.For example, this term includes any disorder, disease or condition that can lead to hyperlipidemia or a condition characterized by abnormally elevated levels of any or all lipids and / or lipoproteins in blood.This term refers to inherited diseases such as familial hypertriglyceridemia, or acquired diseases such as disorders acquired as a result of dietary habits or taking certain drugs. Exemplary lipid metabolism disorders include, but are not limited to, atherosclerosis, hyperlipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcoholic hypertriglyceridemia, beta-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrome, familial chyloemia, apoE deficiency or resistance, LPL deficiency or reduced function, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, and xanthomatosis (subcutaneous cholesterol deposition).

[0080] Cardiovascular diseases associated with lipid metabolism disorders are also considered to be "lipid metabolism disorders" as defined herein. These diseases can include coronary artery disease (also called ischemic heart disease), inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, and stroke.

[0081] Weight-related disorders, as defined herein, are also considered "lipid metabolism disorders." Such disorders may include obesity, metabolic syndrome, including independent components of metabolic syndrome (e.g., central obesity, FBG / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of regaining weight after weight loss.

[0082] Glycemic disorders are also considered "lipid metabolism disorders" as defined herein. Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome associated with insulin resistance. Other exemplary lipid metabolism disorders may also include kidney transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogen storage disease type I, and Addison's disease.

[0083] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject suffering from a lipid metabolism disorder, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the manner in which the agent is administered, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0084] As used herein, a "prophylactically effective amount" is intended to include an amount of an iRNA that, when administered to a subject suffering from a lipid metabolism disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of later-onset disease. A "prophylactically effective amount" may vary depending on the iRNA, the manner in which the agent is administered, the risk of contracting the disease, and the medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0085] A "therapeutically effective amount" or a "prophylactically effective amount" also includes an amount of an RNAi agent that produces a desired local or systemic effect with a reasonable benefit / risk ratio applicable to any treatment. The iRNAs used in the methods of the invention can be administered in an amount sufficient to obtain a reasonable benefit / risk ratio applicable to such treatment.

[0086] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human and animal subjects without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-risk ratio.

[0087] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, talc, magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound to a subject from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials which may serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) celluloses and their derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) propylene glycol, and the like. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free distilled water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0088] The term "sample" as used herein includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be from specific organs, parts of organs, or fluids or cells in those organs. In certain embodiments, samples can be from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells in the liver, e.g., hepatocytes). In certain embodiments, a "sample from a subject" refers to blood or plasma obtained from a subject.

[0089] II. iRNAs of the Invention Described herein is an iRNA that inhibits the expression of ANGPTL3 gene.In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of ANGPTL3 gene in a cell, such as a cell in a subject, for example, a mammal (e.g., a human suffering from lipid metabolism disorder, such as familial hyperlipidemia).The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed during the expression of ANGPTL3 gene, and the region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the ANGPTL3 gene, the iRNA inhibits expression of the ANGPTL3 gene (e.g., a human, primate, non-primate, or avian ANGPTL3 gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA) methods, or by protein-based methods, such as, for example, by immunofluorescence analysis using Western blot or flow cytometry techniques.

[0090] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a region of complementarity that is substantially complementary to the target sequence, generally completely complementary. The target sequence can be derived from the sequence of the mRNA formed during the expression of ANGPTL3 gene. The other strand (sense strand) comprises a region that is complementary to the antisense strand, such that the two strands hybridize to form a double-stranded structure when suitable conditions are combined. As described elsewhere herein and as known in the art, the complementary sequence of dsRNA can also be included as a self-complementary region of one nucleic acid molecule, rather than being on a separate oligonucleotide.

[0091] Generally, the double-stranded structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-2 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated as part of the invention.

[0092] Similarly, the region of complementarity to the target sequence may be 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 1 9-29, 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 nucleotides in length. Ranges and lengths intermediate to the above listed ranges and lengths are also contemplated as part of the invention.

[0093] In some embodiments, the dsRNA is about 15 to about 20 nucleotides long, or about 25 to about 30 nucleotides long. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21 to 23 nucleotides can serve as a substrate for Dicer. As those skilled in the art will also recognize, the region of the RNA targeted for cleavage is most often a portion of a larger RNA molecule, often an mRNA molecule. Where relevant, a "portion" of the mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0094] The double-stranded region may be a primary functional portion of the dsRNA, e.g., about 9-36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-34 3, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-2 4, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 1 Those skilled in the art will also recognize that the double-stranded region may be 9-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Here, in one embodiment, to the extent that the RNA molecule or the complex of RNA molecules having a double-stranded region of more than 30 base pairs is processed into a functional double-strand of, for example, 15-30 base pairs that targets the desired RNA for cleavage, it is dsRNA. Thus, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting the expression of ANGPTL3 is not generated in a target cell by cleavage of a larger dsRNA.

[0095] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. A dsRNA having at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to its blunt-ended counterpart. The nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhang nucleotide may be present on the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0096] dsRNA can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, Inc.), as further described below.

[0097] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds 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 easily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0098] 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 2, 3, 7, 8, 9 and 10, and the corresponding antisense strand of the sense strand is selected from the group of sequences shown in Tables 2, 3, 7, 8, 9 and 10. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the mRNA produced during the expression of the ANGPTL3 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, where one oligonucleotide is represented as the sense strand in Tables 2, 3, 7, 8, 9 and 10, and the second oligonucleotide is represented as the corresponding antisense strand of the sense strand in Tables 2, 3, 7, 8, 9 and 10. In one embodiment, the substantially complementary sequences of the dsRNA are comprised in separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are comprised in one oligonucleotide.

[0099] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of about 20-23 base pairs, e.g., 21 base pairs, have been found to be particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others skilled in the art have found that shorter or longer RNA double-stranded structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above embodiments, depending on the nature of the oligonucleotide sequences shown in Tables 2, 3, 7, 8, 9 and 10, the dsRNAs described herein may include at least one strand of a minimum length of 21 nucleotides. It can be reasonably expected that a shorter duplex having one of the sequences in Tables 2, 3, 7, 8, 9 and 10, minus a small number of nucleotides at one or both ends, can be similarly effective compared to the above dsRNA.Therefore, it is considered within the scope of the present invention that the dsRNA has a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one of the sequences in Tables 2, 3, 7, 8, 9 and 10, and the ability to inhibit the expression of ANGPTL3 gene differs from the dsRNA having the complete sequence by about 5, 10, 15, 20, 25 or 30% inhibition or less.

[0100] Furthermore, the RNAs shown in Tables 2, 3, 7, 8, 9 and 10 identify sites in the ANGPTL3 transcript that are susceptible to RISC-mediated cleavage. Thus, the present invention further features an iRNA that targets within one of these sites. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA induces cleavage of the transcript anywhere within that specific site. Such an iRNA will generally comprise at least about 15 contiguous nucleotides from one of the sequences shown in Tables 2, 3, 7, 8, 9 and 10 linked to additional nucleotide sequences taken from regions adjacent to the selected sequence in the ANGPTL3 gene.

[0101] Target sequences are generally about 15-30 nucleotides in length, although the suitability of specific sequences within this range to direct cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for the identification of optimal target sequences for any given gene target, an empirical approach can also be taken in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed literally or figuratively (including, for example, in silico) over the target RNA sequence to identify sequences within a size range that may serve as target sequences. The next potential target sequence can be identified by gradually moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence location until a complete set of possible sequences has been identified for any given target size selected. This process, along with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify sequences that function optimally, can identify RNA sequences that mediate the best inhibition of expression of the target gene when targeted with an iRNA agent. Thus, for example, while the sequences identified in Tables 2, 3, 7, 8, 9 and 10 represent effective target sequences, it is believed that further optimization of inhibitory efficiency may be achieved by gradually "shifting the window" one nucleotide upstream or downstream of the given sequence to identify sequences with comparable or better inhibitory properties.

[0102] It is further contemplated that further optimization can be achieved, for example, by systematically adding or removing nucleotides to generate longer or shorter sequences for any sequence identified in Tables 2, 3, 7, 8, 9, and 10, and testing the sequences generated by moving longer or shorter size windows up or down the target RNA from that point. This approach to generate new candidate targets can also be coupled with testing the effectiveness of iRNAs based on those target sequences in inhibition assays known in the art and / or described herein, further improving the efficiency of inhibition. Furthermore, such optimized sequences can be adjusted, for example, by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or described herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, improving transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

[0103] The iRNA described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNA described herein contains three or less mismatches. When the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the area of ​​mismatch is not located in the center of the region of complementarity. When the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the mismatch is limited to within the last 5 nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 23 nucleotide iRNA agent, the strand complementary to a region of the ANGPTL3 gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an iRNA containing mismatches with the target sequence is effective in inhibiting the expression of the ANGPTL3 gene. Considering the effectiveness of an iRNA with mismatches in inhibiting the expression of the ANGPTL3 gene is important, especially when a particular region of complementarity in the ANGPTL3 gene is known to have polymorphic sequence variation in the population.

[0104] III. Modified iRNAs of the Invention In one embodiment, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to improve stability or other beneficial properties. The nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acids chemistry," Beaucage, SLet al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5'-end modifications (phosphorylation, conjugates, inverted linkages) or 3'-end modifications (conjugates, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stable bases, unstable bases, or bases that base-pair with a wide range of partners, removal of bases (non-basic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or replacement of sugars; and / or backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones or do not contain natural internucleoside linkages. RNAs with modified backbones include, in particular, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in the internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, modified iRNAs have a phosphorus atom in their internucleoside backbone.

[0105] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with inverted polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0106] Representative United States patents which teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,32 Specification No. 1,131; Specification No. 5,399,676; Specification No. 5,405,939; Specification No. 5,453,496; Specification No. 5,455,233; Specification No. 5,466,677; Specification No. 5,476,925 Specification No. 5,519,126; Specification No. 5,536,821; Specification No. 5,541,316; Specification No. 5,550,111; Specification No. 5,563,253; Specification No. 5,571,799; Specification No. 5,587 ,361 Specification; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209 ; 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, Nos. 6,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0107] Modified RNA backbones that do not contain internal phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, or mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S and CH2 constituent moieties.

[0108] Representative United States patents which teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; Nos. 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0109] In other embodiments, suitable RNA mimics are contemplated for use in iRNA, where both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide unit, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such RNA mimic that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0110] Certain embodiments featured in the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly those of the aforementioned U.S. Patent No. 5,489,677, such as --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (also known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--), and the aforementioned amide backbones of U.S. Patent No. 5,602,240. In certain embodiments, RNAs featured herein have the morpholino backbone structure of the aforementioned U.S. Patent No. 5,034,506.

[0111] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein, may include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(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 In another embodiment, the dsRNA comprises at the 2' position a C1 to C2 CH3)]2, where n and m are from 1 to about 10. 10The modifications include one of lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacokinetic properties of iRNA, or group that improves the pharmacodynamic properties of iRNA, and other substituents with similar properties. In certain embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the Examples herein below.

[0112] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in a 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents which teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,81 Nos. 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, several of which are commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0113] iRNAs may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-aminouracil (pseudouracil), 5 ... Other synthetic and natural nucleobases include 5-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. The 5-methylcytosine substitution has been shown to increase nucleic acid duplex 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) and is an exemplary base substitution, especially when combined with a 2'-O-methoxyethyl sugar modification.

[0114] Representative United States patents which teach the preparation of some of the above-mentioned modified nucleobases as well as other modified nucleobases include, but are not limited to, the above-mentioned U.S. Patents 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,1 Specification No. 21, Specification No. 5,596,091; Specification No. 5,614,617; Specification No. 5,681,941; Specification No. 5,750,692; Specification No. 6, Specification No. 015,886; Specification No. 6,147,200; Specification No. 6,166,197; Specification No. 6,222,025; Specification No. 6,235,887 Nos. 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0115] The RNA of the iRNA can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, which contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo structural conformation. The addition of locked 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).

[0116] Representative United States patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are incorporated herein by reference.

[0117] Potentially stable modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO 2011 / 005861.

[0118] IV. iRNA bound to ligand Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include, but are not limited to, cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86:6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), thiocholesterol (Oberhauser et al., (1992) Nucl. Acids ... Res., 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids, 75:49-54). Res., 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmityl moieties (Mishra et al., (1995) Biochim. Biophys.Acta, 1264:229-237), or lipid moieties such as octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0119] In one embodiment, a ligand alters the distribution, targeting, or life span of an iRNA agent into which it is incorporated. In a preferred embodiment, a ligand provides improved affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a compartment of a cell or organ), tissue, organ, or region of the body, for example, as compared to a species lacking such a ligand. Preferred ligands are not involved in the pairing of the two strands in a double-stranded nucleic acid.

[0120] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymers, poly(L-lactide-co-glycolide) copolymers, divinyl ether-maleic anhydride copolymers, N-(2-hydroxypropyl) methacrylamide copolymers (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethanes, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazines. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidines, protamines, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0121] The ligand may also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody that binds to a specific cell type, such as a renal cell. The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0122] Other examples of ligands include dyes, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid (cholenic acid), acid), dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugates, Eu3+ tetraazamacrocycle conjugates), dinitrophenyl, HRP, or AP.

[0123] A ligand can be a protein, e.g., a glycoprotein, or a peptide, e.g., a co-ligand, or an antibody, e.g., a molecule with specific affinity for an antibody that binds to a particular cell type, such as a hepatocyte. Ligands can also include hormones and hormone receptors. Ligands can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. A ligand can be, for example, lipopolysaccharide, an activator of MAP kinase 38, or an activator of NF-κB.

[0124] The ligand can be a substance, e.g., a drug, that can enhance uptake of the iRNA agent into the cell, e.g., by disrupting the cytoskeleton, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0125] In some embodiments, the ligands attached to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing some phosphorothioate linkages are also known to bind to serum proteins, and therefore short oligonucleotides, for example, oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable for the present invention as ligands (e.g., PK-modulating ligands). Additionally, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0126] The ligand-conjugated oligonucleotides of the invention can be synthesized by the use of oligonucleotides bearing reactive pendant functional groups, such as those derived from the attachment of a binding molecule to the oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands having a binding moiety attached thereto.

[0127] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely produced by the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may be used in addition or instead. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0128] In the ligand-conjugated oligonucleotides and ligand molecules having sequence-specifically bound nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled in a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already have a binding moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0129] When using a nucleotide conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is usually completed before the ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates in addition to commercially available standard and non-standard phosphoramidites commonly used in oligonucleotide synthesis.

[0130] A. Lipid Conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecule is preferably bound to serum protein, for example, human serum albumin (HSA). HSA-binding ligand allows the distribution of the conjugate to target tissues in the body, for example, non-renal target tissues. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, neproxin or aspirin can be used. Lipid or lipid-based ligand can be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum protein, for example, HSA.

[0131] Lipid-based ligand can be used to inhibit, for example, control, the binding of conjugate to target tissue.For example, lipid or lipid-based ligand that binds stronger to HSA is less likely to be targeted to kidney, and therefore less likely to be removed from the body.Lipid or lipid-based ligand that binds weaker to HSA can be used to target conjugate to kidney.

[0132] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity such that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that the HSA-ligand binding cannot be reversed.

[0133] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate is preferably distributed to the kidney. Other moieties that target renal cells can be used in place of or in addition to the lipid-based ligand.

[0134] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by, e.g., malignant or non-malignant unwanted cell proliferation, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, such as hepatocytes. Also included are HSA and low density lipoprotein (LDL).

[0135] B. Cell-penetrating agents In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic and lipophobic phase.

[0136] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to an iRNA agent can affect the pharmacokinetic distribution of the iRNA, such as by improving cellular recognition and uptake. The peptide or peptidomimetic portion can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0137] The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12) have been shown to be capable of functioning as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries, or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., 2003). al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic linked to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below can be used.

[0138] The RGD peptide for use 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 to specific tissues.RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimetics.In addition to RGD, other moieties that target integrin ligands may be used.Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0139] A "cell-penetrating peptide" is capable of penetrating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A peptide that penetrates a microbial cell can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). A cell-penetrating peptide can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0140] C. Carbohydrate Conjugates In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for delivery of nucleic acids in vivo, as described herein, and the compositions are suitable for therapeutic use in vivo. As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each of which is bound to an oxygen, nitrogen, or sulfur atom; or a compound that has as a part thereof a carbohydrate moiety composed of one or more monosaccharide units, each of which has at least six carbon atoms (which may be linear, branched, or cyclic), each of which is bound to an oxygen, nitrogen, or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 and above (e.g., C5, C6, C7, or C8); di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0141] In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. [ka] and other N-acetylgalactosamines.

[0142] In another embodiment, the carbohydrate conjugate used in the compositions and methods of the invention comprises: [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0143] Further representative carbohydrate conjugates for use in the embodiments described herein include, but are not limited to, [ka] (Formula XXIII), wherein when one of X or Y is an oligonucleotide, the other is hydrogen.

[0144] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands, as described above, including, but not limited to, a PK modulator and / or a cell penetrating peptide.

[0145] D. Linker In some embodiments, the conjugates or ligands described herein may be attached to the iRNA oligonucleotide using a variety of linkers, which may be cleavable or non-cleavable.

[0146] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, for example, covalently bonding the two parts of a compound. Linkers are typically direct bonds, or atoms such as oxygen or sulfur, units such as NR, C(O), C(O)NH, SO, SO, SONH, or units such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhetero(herero)cyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkene and nyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheter(herero)aryl (wherein one or more methylenes may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic); where R8 is hydrogen, acyl, aliphatic or substituted aliphatic.In one embodiment, the linker consists of about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0147] A cleavable linking group is one that is sufficiently stable outside a cell, but is cleaved after entry into a target cell to release the two moieties that the linker holds together. In preferred embodiments, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, or at least about 100 times faster inside the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0148] The conjugable linking group is sensitive to a cleaving agent, such as pH, redox potential, or the presence of a degradable molecule. In general, the cleaving agent is found to be widespread or at a higher level or activity inside cells compared to serum or blood. Examples of such degrading agents include: oxidizing or reducing enzymes present in cells, or redox agents selected for a specific substrate or with no substrate specificity, including reducing agents such as mercaptans, which can degrade redox-cleavable linking groups by reduction; esterases; agents that can create endosomes or acidic environments, such as pH below 5; enzymes that can act as general acids, thereby hydrolyzing or degrading acid-cleavable linking groups, peptidases (which can be substrate specific), and phosphatases.

[0149] Cleavable linking groups such as disulfide bonds can be pH sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into a desired compartment of the cell.

[0150] The linker may contain a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell to be targeted. For example, a liver-targeting ligand may be linked to a cationic lipid via a linker that contains an ester group. Since liver cells are rich in esterases, this linker will be cleaved more efficiently in liver cells compared to cell types that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

[0151] Linkers containing peptide bonds can be used in targeting cell types rich in peptidases, such as hepatocytes and synovial cells.

[0152] In general, the suitability of a candidate cleavable binding group can be evaluated by testing the ability of a degrading agent (or degrading condition) to cleave the candidate binding group. It may also be desirable to test the ability of the candidate cleavable binding group to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first condition and a second condition can be determined, the first condition being selected to indicate cleavage in target cells and the second condition being selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. This evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform an initial evaluation in a cell-free or culture condition and confirm with further evaluation in the animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster within cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0153] i. Cleavable redox linking group In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved after reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one may look to the methods described herein. For example, the 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 would be observed in cells, for example, target cells. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In one such case, the candidate compound is cleaved at most about 10% in blood. In another embodiment, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster inside a cell (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0154] ii. Phosphate-based cleavable linking groups In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group that is cleaved by an agent that degrades or hydrolyzes the phosphate group. One example of an agent that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. Preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0155] iii. Acid-cleavable linking groups In another embodiment, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less) or by an agent such as an enzyme that can act as a general acid. Within a cell, certain low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is where the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0156] iv. Ester-Based Cleavable Linking Groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amylases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O-, or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0157] v. Peptide-Based Cleavable Linkers In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes, such as peptidases and proteases, in cells. An example of a peptide-based cleavable linking group is a peptide bond formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C(O)NH-). An amide group 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 cleaving 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 has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0158] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrates conjugated to linkers of the compositions and methods of the present invention include: [ka] [ka] [ka] When one of X and Y is an oligonucleotide, the other is hydrogen.

[0159] In certain embodiments of the compositions and methods of the invention, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0160] In one embodiment, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XXXI) to (XXXIV): [ka] wherein q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently for each occurrence represent 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C are each, independently for each occurrence, absent, 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 is independently for each occurrence absent, alkylene, or substituted alkylene, where one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5Care each independently absent for each occurrence, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each, independently for each occurrence, is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives, such as those of formula (XXXV), are particularly useful in conjunction with RNAi agents to inhibit expression of target genes: [ka] In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative.

[0161] Examples of suitable divalent and trivalent branched linking groups for conjugation to GalNAc derivatives include, but are not limited to, the structures cited above as Formulas II_VII, XI, X, and XIII.

[0162] Representative patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,80 2; U.S. Patent No. 5,138,045; U.S. Patent No. 5,414,077; U.S. Patent No. 5,486,603; U.S. Patent No. 5,512,439; U.S. Patent No. 5,578,718; U.S. Patent No. 5,608,046; U.S. Patent No. 4,587,044; U.S. Patent No. 4,605,735; U.S. Patent No. 4,667,025; U.S. Patent No. 4,762,779; U.S. Patent No. 4,789,737; U.S. Patent No. 4,824,941; U.S. Patent No. 4,835,2 63; U.S. Patent No. 4,876,335; U.S. Patent No. 4,904,582; U.S. Patent No. 4,958,013; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,245,022; U.S. Patent No. 5,254,469; U.S. Patent No. 5,258,506; U.S. Patent No. 5,262, 536; U.S. Patent No. 5,272,250; U.S. Patent No. 5,292,873; U.S. Patent No. 5,317,098; U.S. Patent No. 5,371,241, U.S. Patent No. 5,391,723; U.S. Patent No. 5,416,203, U.S. Patent No. 5,451,463; U.S. Patent No. 5,510,475; U.S. Patent No. 5,512,667; U.S. Patent No. 5,514,785; U.S. Patent No. 5,565,552; U.S. Patent No. 5,567,810; U.S. Patent No. 5,574,142;Nos. 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0163] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the above modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0164] A "chimeric" iRNA compound or "chimera" in the context of the present invention is an iRNA compound, preferably a dsRNA, that comprises two or more chemically distinct regions, each composed of at least one monomer unit, i.e., in the case of a dsRNA compound, a nucleotide. These iRNAs usually comprise at least one region, in which the RNA is modified to confer to the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, comparable results are often obtained with shorter iRNAs compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can conveniently be detected by gel electrophoresis and, optionally, associated nucleic acid hybridization techniques known in the art.

[0165] In some cases, the RNA of an iRNA may be modified by a non-ligand group. Several non-ligand molecules have been attached to an iRNA to improve the activity, cellular distribution or cellular uptake of the iRNA, and procedures for making such attachments are available in the scientific literature.Such non-ligand moieties include cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative US patents that teach the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions of the sequence. The amino group is then reacted with the conjugated molecule using a suitable coupling or activating agent. The conjugation reaction can be carried out with the RNA still bound to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC usually results in a pure conjugate.

[0166] IV. Delivery of iRNA of the Invention Delivery of the iRNA of the present invention to a cell, e.g., a cell in a subject, such as a human subject (e.g., a subject in need of an iRNA, such as a subject suffering from a lipid metabolism disorder), can be accomplished in a number of different ways. For example, delivery can be accomplished by contacting a cell with an iRNA of the present invention, either in vitro or in vivo. In vivo delivery can also be accomplished directly by administering a composition comprising an iRNA, e.g., a dsRNA, to the subject. Alternatively, in vivo delivery can be accomplished indirectly, by administering one or more vectors that encode and direct the expression of the iRNA. Examples of these alternatives are described further below.

[0167] In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with the iRNA of the present invention (see, e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to be considered for delivering an iRNA molecule include, for example, the biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. Non-specific effects of iRNA can be minimized by local administration, e.g., by direct injection or implantation into the tissue, or by administering the formulation locally. Local administration at the treatment site can maximize the local concentration of the agent, limit exposure of the agent to systemic tissues that may be adversely affected by or degrade the agent, and allow for a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al. (2003) Mol. Vis. 9:210-216) have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice can reduce tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and extend survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523).RNA interference has been demonstrated by localized delivery to the central nervous system via direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs via intranasal administration (Howard, KA. et al. al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). To administer iRNA systemically for the treatment of disease, the RNA can be modified or delivered using a drug delivery system; both methods serve to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutical carrier can also allow for the targeting of iRNA compositions to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol, which improves cellular uptake and prevents degradation. For example, iRNA against ApoB conjugated to a lipophilic cholesterol moiety was administered systemically to mice, resulting in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of iRNA to an aptamer 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 alternative embodiments, the iRNA can be delivered using a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system. The positively charged cationic delivery system promotes the binding of the iRNA molecule (which is negatively charged) and also improves the interaction with the negatively charged cell membrane, allowing for efficient uptake of the iRNA by the cell. The cationic lipids, dendrimers, or polymers can be bound to the iRNA or induced to form vesicles or micelles (see, for example, Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116) that encapsulate the iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of one of ordinary skill in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111-114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethylenimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of time). print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D. A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In certain embodiments, the iRNA is complexed with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrin can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

[0168] A. Vector-Encoded iRNA of the Invention iRNAs targeting the ANGPTL3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A. et al., International PCT Publication No. WO 00 / 22113, Conrad, International PCT Publication No. WO 00 / 22114, and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a matter of hours to weeks) or sustained (weeks to months or longer), depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. The transgene can also be constructed to allow it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

[0169] The individual strand or strands of the iRNA can be transcribed from a promoter in an expression vector. Two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell, where two separate strands are expressed to produce, for example, dsRNA. Alternatively, each individual strand of the dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence to have a stem-loop structure.

[0170] The iRNA expression vector is generally a DNA plasmid or a viral vector. Recombinant constructs for the expression of the iRNA described herein can be produced using expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells. Eukaryotic expression vectors are well known in the art and are available from many commercial sources. Such vectors are usually provided with convenient restriction sites for inserting the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, such as by intravenous or intramuscular administration, by administration to target cells transplanted from the patient and then reintroduced into the patient, or by any other means that allows introduction into the desired target cells.

[0171] The iRNA expression plasmid can be transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of a week or more are also contemplated by the present invention. Successful introduction of the vector into the host cell can be monitored using a variety of known methods. For example, transient transfection can be indicated using a reporter, such as a fluorescent marker, such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using a marker that confers resistance to certain environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance, to the transfected cells.

[0172] 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, Moloney murine leukemia virus, etc.; (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-defective viruses may also be advantageous. Different vectors may or may not integrate into the genome of the cell. The construct may include viral sequences for transfection, as appropriate. Alternatively, the construct may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in the target cell. Other aspects to be considered for vectors and constructs are described further below.

[0173] Vectors useful for delivery of iRNA will contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulatable / inducible expression.

[0174] The expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as blood glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems suitable for controlling the expression of dsRNA in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select the appropriate regulatory / promoter sequence based on the intended use of the iRNA transgene.

[0175] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., (1993) Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to the patient. Further details on retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references showing the use of retroviral vectors in gene therapy are Clowes et al., (1994) J. Clin. Invest. 93:644-651; Kiem et al., (1994) Blood 83:1467-1473; Salmons and Gunzberg, (1993) Human Gene Therapy 4:129-141; and Grossman and Wilson, (1993) Curr. Opin. in Genetics and Devel. 3:110-114. Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0176] Adenoviruses are also contemplated for use in delivering the iRNA of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes, for example, to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia and cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, (1993) Current Opinion in Genetics and Development 3:499-503, provide a review of adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to respiratory epithelia in rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155; Mastrangeli et al., (1993) J. Clin. Invest. 91:225-234; PCT Publication WO 94 / 12649; and Wang et al., (1995) Gene Therapy 2:775-783. AV vectors suitable for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0177] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNA of the invention (Walsh et al., (1993) Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0178] Another viral vector suitable for delivery of the iRNA of the invention is a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, a poxvirus such as an avian pox, e.g., fowlpox or canarypox.

[0179] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be made to target different cells by engineering the vector to express different capsid protein serotypes. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0180] The pharmaceutical preparation of the vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0181] V. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention.In one embodiment, a pharmaceutical composition is provided herein that contains the iRNA described herein and a pharma- ceutical acceptable carrier.The pharmaceutical composition containing the iRNA is useful for treating diseases or disorders associated with the expression or activity of the ANGPTL3 gene, such as lipid metabolism disorders, such as hypertriglyceridemia.

[0182] Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral administration, e.g., by intravenous (IV) or subcutaneous delivery. Another example is a composition formulated for direct delivery to the liver, e.g., by injection into the liver, such as by continuous pump infusion.

[0183] The pharmaceutical composition of the present invention can be administered at a dosage sufficient to inhibit the expression of the ANGPTL3 gene. In general, the suitable dosage of the iRNA of the present invention is in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally in the range of about 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single administration.

[0184] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8.8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8.9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0185] In another embodiment, the dsRNA is about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg , about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about About 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / mg, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m g / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present invention.

[0186] For example, dsRNA may be present at approximately 0..01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 5, 4.6, 4.7, 4.8.4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8.8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8.9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0187] In another embodiment, the dsRNA is about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 ... 0mg / kg, about 30 to about 50mg / kg, about 35 to about 50mg / kg, about 40 to about 50mg / kg, about 45 to about 50mg / kg, about 0.5 to about 45mg / kg, about 0.75 to about 45mg / kg, about 1 to about 45mg / mg, about 1.5 to about 45mg / kb, about 2 to about 45mg / k g, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg mg, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 ~40mg / kg, 15~40mg / kg, 20~40mg / kg, 25~40mg / kg, 25~40mg / kg, 30~40mg / kg, 35~40mg / kg, 0.5~30mg / kg, 0.75~30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.It is administered at a dose of about 5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Intermediate values ​​and ranges of the values ​​described are also intended to be part of the present invention. .

[0188] For example, subjects may have approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3. 9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8.4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 .7, 7.8.7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8.8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8.9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, Treatment amounts of iRNA such as 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg may be administered. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0189] The pharmaceutical composition can be administered once a day, or the iRNA can be administered as two, three or more subdoses at appropriate intervals throughout the day, or even using delivery via continuous infusion or sustained release formulations. In that case, each subdose should contain a correspondingly smaller amount of iRNA to achieve the total daily dosage. The dosage unit can also be formulated for delivery over several days, for example using a conventional sustained release formulation that provides sustained release of the iRNA over a period of several days. Sustained release formulations are well known in the art and can be used with the agents of the invention, as they are particularly useful for delivering agents to specific sites. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0190] The effect of a single dose on ANGPTL3 levels can be long-lasting, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days, or no more than 1, 2, 3, or 4 weeks.

[0191] One of skill in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can affect the dosage and time required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be estimated using conventional methodology or based on in vivo testing using appropriate animal models as described elsewhere herein.

[0192] Advances in mouse genetics have generated many mouse models for the study of various human diseases, such as lipid metabolism disorders, that may benefit from reduced expression of ANGPTL3. Such models can be used for in vivo testing of iRNAs as well as to determine therapeutically effective doses. Suitable mouse models are known in the art and include, for example, obese (ob / ob) mice containing a mutation in the obese (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); mice containing a homozygous knockout of the LDL receptor (LDLR- / - mice; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); diet-induced atherosclerosis mouse model (Ishida et al., (1991) J. Lipid. Res., 32:559-568); and a heterozygous lipoprotein lipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest. 96(6):2555-2568).

[0193] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is required and the site to be treated. Administration can be topical (e.g., by transdermal patch), pulmonary administration, for example, by inhalation or insufflation of powders or aerosols, such as by nebulizers; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous administration, for example, by implantation device; or intracranial administration, for example, by intraparenchymal, intrathecal or intraventricular administration.

[0194] The iRNA can be delivered to target a specific tissue, such as the liver (e.g., hepatocytes of the liver).

[0195] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like may be necessary or desirable. Covered condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the present invention are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleyl phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), anionic (e.g., dimyristoyl phosphatidylglycerol DMPG) and cationic (e.g., dioleyl tetramethylaminopropyl DOTAP and dioleyl phosphatidylethanolamine DOTMA). The iRNAs featured in the present invention can be encapsulated in liposomes or complexed to liposomes, particularly cationic liposomes. Alternatively, the iRNAs can be complexed to lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharma- ceutically acceptable salts thereof.Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0196] A. iRNA formulations containing membrane molecular assemblies iRNA 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, such as one or more bilayers. Liposomes include unilamellar and multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior and usually does not contain the iRNA composition, but in some cases it may. Liposomes are useful for transporting and delivering active ingredients to the site of action. Because the liposomal membrane is similar in structure to biological membranes, when the liposome is attached to a tissue, the liposomal bilayer fuses with the cell membrane bilayer. As the fusion of the liposome and the cell proceeds, the aqueous contents of the interior, including the iRNA, are delivered to the cell, where the iRNA can specifically bind to the target RNA and mediate RNAi. In some cases, the liposomes are also specifically targeted, for example, to direct the iRNA to a particular cell type.

[0197] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent so that micelles are formed with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles containing the lipid components. The cationic groups in the lipids interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposomal formulation of the RNAi agent.

[0198] If necessary, a carrier compound that aids in condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to aid in condensation.

[0199] Methods for generating stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as a component of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described in Felgner, PLet al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; al.,(1979)Biochim.Biophys.Acta 557:9;Szoka et al.,(1978)Proc.Natl.Acad.Sci.75:4194;Mayhew et al.,(1984)Biochim.Biophys.Acta 775:169;Kim et al.,(1983)Biochim.Biophys.Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115: 757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw and extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858: 161. When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., (1984) Biochim. Biophys. Acta 775: 169. These methods are easily adapted to package preparations of RNAi agents into liposomes.

[0200] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are transferred into endosomes. The acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., (1987) Biochem. Biophys. Res. Commun.,, 147, 980-985).

[0201] Liposomes that are pH-sensitive and negatively charged entrap nucleic acids rather than complexing with them. Because both the nucleic acid and the lipid are similarly charged, repulsion occurs rather than complexation. Nevertheless, some nucleic acids are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., (1992) Journal of Controlled Release, 19, 269-274).

[0202] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleyl phosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0203] Other exemplary methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

[0204] Non-ionic liposomal systems, especially those containing non-ionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin.Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) non-ionic liposomal formulations were used to deliver cyclosporine-A to the dermis of mouse skin.The results showed that such non-ionic liposomal systems were effective in promoting the deposition of cyclosporine-A in different layers of the skin (Hu et al (1994). STP Pharma. Sci., 4, 6, 466).

[0205] Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, provide enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) that are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes results from reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223, 42; Wu et al., (1993) Cancer Research, 53, 3765).

[0206] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) report the use of monosialoganglioside G to improve the blood half-life of liposomes. M1 (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G to inhibit the cellular cellular endothelial function of sphingomyelin. M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0207] In one embodiment, cationic liposome is used.Cationic liposome has the advantage that it can fuse with cell membrane.Non-cationic liposome cannot fuse with cell membrane as efficiently, but it can be taken up by macrophage in vivo and can be used to deliver RNAi agent to macrophage.

[0208] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; liposomes can protect the RNAi agents encapsulated in their internal compartment from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0209] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of tissue culture cell membranes, resulting in delivery of RNAi agents (see, e.g., Felgner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0210] A DOTMA analog, 1,2-bis(oleyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously attach to negatively charged cell surfaces, fuse with cell membranes, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are attached by ester rather than ether bonds.

[0211] Other reported cationic lipid compounds include those conjugated to one of two types of lipids and conjugated to a variety of moieties, including, for example, carboxyspermine, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0212] Another cationic lipid conjugate involves derivatization of lipids with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0213] Liposome formulations are particularly suitable for topical administration, and liposomes exhibit several advantages over other formulations.These advantages include the reduced side effects associated with the high systemic absorption rate of the administered drug, the increased accumulation of the administered drug in the desired target, and the ability to administer RNAi agents to the skin.In some implementations, liposomes are used to deliver RNAi agents to epidermal cells and to promote the penetration of RNAi agents into dermal tissues, such as skin.For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been reported (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, RJ and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, RM and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. and See Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0214] Non-ionic liposomal systems, particularly those containing non-ionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin.Non-ionic liposomal formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs to the dermis of mouse skin.Such formulations containing RNAi agents are useful for treating skin diseases.

[0215] Liposomes containing iRNA can be made highly deformable. Such deformability can allow liposomes to penetrate pores smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be made by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing RNAi agents can be delivered, for example, by subcutaneous infection, to deliver RNAi agents to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must penetrate a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to lipid properties, these transferosomes can be self-optimizing (e.g., adaptable to the shape of pores), self-repairing, often reaching their target without fracturing, and often self-loading.

[0216] Other formulations suitable for the present invention are described in U.S. Provisional Patent Application Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008, and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations suitable for the present invention.

[0217] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive as potential drug delivery vehicles. Transfersomes can also be described as lipid droplets that are highly deformable and therefore can easily penetrate into pores smaller than the droplets. Transfersomes can adapt to the environment in which they are used, for example, they are self-optimizing (adapting to the shape of the pores in the skin), self-repairing, often reach their target without fragmentation, and are often self-loading. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0218] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0219] If the surfactant molecule is not ionized, the surfactant is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values ​​range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0220] If surfactant molecule carries negative charge when dissolved or dispersed in water, this surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyltaurates and sulfosuccinates, and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

[0221] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.

[0222] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0223] The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0224] The iRNA for use in the method of the present invention can also be provided as a micelle formulation.A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure, so that all the hydrophobic parts of the molecule face inward, and the hydrophilic parts remain in contact with the surrounding aqueous phase.When the environment is hydrophobic, the reverse arrangement exists.

[0225] Mixed micelle formulations suitable for transdermal delivery contain an aqueous solution of the siRNA composition, an alkali metal ion, 22 The mixed micelles can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, hyaluronic acid pharmaceutically acceptable salts, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polidocanol alkyl ether and its analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compounds may be added simultaneously with or after the addition of alkali metal alkyl sulfates. Mixed micelles can be formed by virtually any type of mixing of components, but are formed by vigorous mixing to provide smaller size micelles.

[0226] In one method, a first micelle composition is prepared containing an siRNA composition and at least an alkali metal alkyl sulfate. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, the micelle composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulfate, and at least one of the micelle-forming compounds, followed by adding the remaining micelle-forming compounds under vigorous mixing.

[0227] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle forming components. An isotonicity agent, such as glycerin, may also be added after formation of the mixed micelle composition.

[0228] For delivery of the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, which is filled with a propellant. The propellant, under pressure, is in liquid form in the dispenser. The ratio of the components is adjusted so that there is one aqueous phase and one propellant phase, i.e., one phase. If there are two phases, the dispenser must be shaken before dispensing a portion of the contents, for example, by a metered valve. The medicinal dose is sprayed from the metered valve in the form of a fine spray.

[0229] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0230] The specific concentrations of the essential components can be determined by relatively simple experimentation. For absorption via the oral cavity, it is often desirable to increase the dosage, for example at least by a factor of two or three, over that for injection or administration via the gastrointestinal tract.

[0231] B. Nucleic acid lipid particles The iDNA or APOC3 dsRNA of the present invention may be fully encapsulated in lipid formulations to form, for example, SPLPs, pSPLPs, SNALPs, or other nucleic acid-lipid particles. The term "SNALP" as used herein refers to a stable nucleic acid-lipid particle that includes SPLPs. The term "SPLP" as used herein refers to a nucleic acid-lipid particle that includes plasmid DNA encapsulated in lipid vesicles. SNALPs and SPLPs typically include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs and SPLPs have an extended circulation life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them extremely useful for systemic application. SPLPs include "pSPLPs," which include encapsulated condensing agent-nucleic acid complexes as shown in PCT Publication No. WO 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, the nucleic acid, when present in the nucleic acid-lipid particles of the present invention, is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. No. 5,976,567; U.S. Pat. No. 5,981,501; U.S. Pat. No. 6,534,484; U.S. Pat. No. 6,586,410; U.S. Pat. No. 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120 and PCT Publication WO 96 / 40964.

[0232] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above ranges are also considered to be part of the invention.

[0233] 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-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-di Methylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof. , (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0234] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0235] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mol percent) with a particle size of 63.0±20 nm and a 0.027 siRNA / lipid ratio.

[0236] Ionic / non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleyl-phosphatidylethanolamine 4-(N The lipid may be an anionic or neutral lipid, including 1-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid, when cholesterol is included, may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0237] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents particle aggregation can be 0 mol% to about 20 mol%, or 2 mol% of the total lipid present in the particle.

[0238] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol %, or about 48 mol % of the total lipid present in the particle.

[0239] In one embodiment, lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Respective stock solutions in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml, PEG-Ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-Ceramide C16 stock solutions can then be combined, 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 ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles usually form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using, for example, a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged, for example, with phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. [ka]

[0240] LNP01 formulations are described, for example, in International Application Publication No. WO 2008 / 042973, which is incorporated herein by reference.

[0241] Further exemplary lipid-dsRNA formulations are described in the table below.

[0242] [Table 1]

[0243] [Table 2]

[0244] [Table 3]

[0245] DSPC: Distearoylphosphatidylcholine DPPC: Dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000) Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminepropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0246] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366, filed January 29, 2009; U.S. Provisional Patent Application No. 61 / 156,851, filed March 2, 2009; U.S. Provisional Patent Application No. 61 / 228,373, filed July 24, 2009; U.S. Provisional Patent Application No. 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0247] Formulations containing MC3 are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed Jun. 10, 2010, the entire contents of which are incorporated herein by reference.

[0248] Formulations containing ALNY-100 are described, for example, in International Patent Application No. PCT / US09 / 63933, filed Nov. 10, 2009, which is incorporated herein by reference.

[0249] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0250] Synthesis of ionic / cationic lipids Any of the compounds, e.g., cationic lipids, used in the nucleic acid-lipid particles of the present invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless otherwise indicated.

[0251] "Alkyl" means a straight or branched, non-cyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched chain alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0252] "Alkenyl" refers to an alkyl as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0253] "Alkynyl" means any alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative straight and branched chain alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0254] "Acyl" means any alkyl, alkenyl, or alkynyl group in which the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0255] "Heterocycle" means a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocycle that is either saturated, unsaturated, or aromatic and contains one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be bonded via any heteroatom or carbon atom. Heterocycle includes heteroaryl as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0256] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle," when substituted, mean that at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, -CN, -ORx, -NRxRy, -NRxC(=O)Ry, -NRxSORy, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy, where n is 0, 1 or 2, and Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORx, heterocycle, -NRxRy, -NRxC(=O)Ry, -NRxSORy, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy.

[0257] "Halogen" means fluoro, chloro, bromo and iodo.

[0258] In some embodiments, the method of the present invention may require the use of a protecting group. Protecting group methods are well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, TW et al., Wiley-Interscience, New York City, 1999). Briefly, a protecting group in the context of the present invention is any group that reduces or eliminates the undesired reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during a particular reaction and then removed to reveal the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the undesired reactivity of an alcohol functional group. A protecting group can be added and removed using techniques well known in the art.

[0259] Synthesis of Formula A In one embodiment, the nucleic acid-lipid particles of the present invention have Formula A: [ka] Formulated with cationic lipid of formula A, wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, R3 and R4 are independently lower alkyl, or R3 and R4 can be taken together to form an optionally substituted heterocycle.In some embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane).Generally, the lipid of formula A above can be prepared by the following reaction scheme 1 or 2, where all substituents are as defined above unless otherwise indicated.

[0260] Scheme 1 [ka] Lipids A, where R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 can be taken together to form an optionally substituted heterocycle, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 gives ketal 3. Treatment of ketal 3 with amine 4 gives lipids of formula A. Lipids of formula A can be converted to the corresponding ammonium salts using organic salts of formula 5, where X is an anionic counterion selected from halogen, hydroxide, phosphate, sulfate, and the like.

[0261] Scheme 2 [ka] Alternatively, the starting material ketone 1 can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 6 and 7 gives ketone 1. Conversion of ketone 1 to the corresponding lipid of formula A is depicted in Scheme 1.

[0262] Synthesis of MC3 The preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid, followed by dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent was removed on a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. Fractions containing the purified product were combined. The solvent was removed to give a colorless oil (0.54 g).

[0263] Synthesis of ALNY-100 The synthesis of ketal 519 [ALNY-100] was carried out according to Scheme 3: [ka]

[0264] Synthesis of 515 To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml of anhydrous THF in a two-necked round-bottom flask (1 L), a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF was slowly added at 0° C. under nitrogen atmosphere. After complete addition, the reaction mixture was allowed to warm to room temperature and then heated to reflux for 4 h. The progress of the reaction was monitored by TLC. After completion of the reaction (by TLC), the mixture was cooled to 00° C. and quenched by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 h and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. The volatiles were removed under reduced pressure to give the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1 H-NMR (DMSO, 400MHz): δ = 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66~2.60 (m, 2H), 2.50~2.45 (m, 5H).

[0265] Synthesis of 516 To a stirred solution of compound 515 in 100 mL of dry DCM in a 250 mL two-neck round bottom flask, NEt3 was added (37.2 mL, 0.2669 mol) and cooled to 0 °C under nitrogen atmosphere. After slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM, the reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC), the mixture was washed successively with 1N HCl solution (1 x 100 mL) and saturated NaHCO3 solution (1 x 50 mL). The organic layer was then dried over anhydrous Na2SO4 and the solvent was evaporated to give the crude material, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11 g (89%). 1 H-NMR (CDCl3, 400MHz): δ=7.36~7.27(m,5H), 5.69(s,2H), 5.12(s,2H), 4.96(br.,1H)2.74(s,3H), 2.60(m,2H), 2.30~2.25(m,2H). LC-MS[M+H]-232.3(96.94%).

[0266] Synthesis of 517A and 517B Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of acetone and water (10:1) in a 500 mL one-neck round-bottom flask, to which N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added followed by 4.2 mL of a 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (about 3 h), the mixture was quenched by the addition of solid Na2SO3 and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 x 100 mL), followed by saturated NaHCO3 (1 x 50 mL) solution, water (1 x 30 mL) and finally brine (1 x 50 mL). The organic phase was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. Silica gel column chromatography purification of the crude material gave a mixture of diastereomers, which were separated by preparative HPLC. Yield:-6 g of crude 517A-peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

[0267] Synthesis of 518 Using a procedure similar to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ = 7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

[0268] General procedure for the synthesis of compound 519 A solution of compound 518 (1 equiv.) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH in THF (1 M, 2 equiv.). After complete addition, the mixture was heated at 40° C. for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4, then filtered through Celite and reduced to an oil. Column chromatography afforded pure 519 (1.3 g, 68%), which was obtained as a colorless oil. 13 C NMR δ = 130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; electrospray MS (+ve): C 44 H 80 Molecular weight (M+H) for NO2 calculated 654.6, found 654.6.

[0269] Formulations prepared by either standard or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. The formulation should be a whitish translucent solution without aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). The particles should be about 20-300 nm in size, for example 40-100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, and the fraction entrapped, are estimated using a dye exclusion assay. Samples of the formulated dsRNA may be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disintegrating surfactant, for example 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant against a standard curve. The percentage entrapped is determined by subtracting the "free" dsRNA content (measured by the signal in the absence of surfactant) from the total dsRNA content. The percentage of entrapped dsRNA is typically >85%. For SNALP formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The preferred range is typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0270] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or mini-tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desired. In some embodiments, oral formulations are those in which the dsRNA characterizing the invention is administered with one or more permeation enhancer surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride, or pharma- ceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as fatty acid / salts in combination with bile acids / salts. One exemplary combination is the sodium salts of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The DsRNA characterizing the present invention can be delivered orally in granular form, including spray-dried particles, or complexed to form micro- or nanoparticles.dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, ... Oral formulations for dsRNA and their formulations are described in U.S. Pat. No. 6,887,906, U.S. Patent Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0271] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharma- ceutically acceptable carriers or excipients.

[0272] The pharmaceutical compositions of the present invention include, but are not limited to, liquid, emulsion, and liposome-containing formulations.These compositions can be made from a variety of components, including, but are not limited to, preformed liquid, self-emulsifying solid, and self-emulsifying semisolid.When treating liver disease, such as hepatocarcinoma, liver-targeting formulations are particularly preferred.

[0273] The pharmaceutical preparation of the present invention, which can be conveniently present in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing active ingredient into association with pharmaceutical carriers or excipients. In general, the preparation is prepared by uniformly and intimately bringing active ingredient into association with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0274] The compositions of the present invention may be formulated into any of a number of possible dosage forms, including but not limited to tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further include substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also include a stabilizer.

[0275] C. Further Formulations i. Emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets, usually with diameters exceeding 0.1 μm (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and (see Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems that contain two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w) types. When the aqueous phase is finely divided and dispersed as minute droplets into a bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to the dispersed phase and active drug, which may be present as a solution in the aqueous phase, the oil phase, or as a separate phase itself. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions may be multiple emulsions consisting of three or more phases, for example, in the case of oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages that simple binary emulsions do not offer. Multiple emulsions in which individual oil droplets of an o / w emulsion enclose small water droplets constitute w / o / w emulsions. Similarly, a system of oil droplets enclosed in globules of water stabilized in a continuous phase of oil provides an o / w / o emulsion.

[0276] Emulsions are characterized by having little or no thermodynamic stability.In many cases, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase and is maintained in this form through the means of emulsifiers or through the viscosity of the formulation.Either of the phases of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams.Other means of stabilizing emulsions include the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0277] Synthetic surfactants, also known as surface active agents, find wide applicability in the formulation of emulsions and are reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199). Surfactants are usually amphiphilic, comprising a hydrophilic portion and a hydrophobic portion. The ratio of hydrophilicity to hydrophobicity of surfactant is called hydrophilic / lipophilic balance (HLB), which is a valuable tool for classifying and selecting surfactants when preparing formulations.Surfactants can be classified into different types based on the nature of their hydrophilic groups, namely nonionic, anionic, cationic and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285).

[0278] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases, such as anhydrous lanolin and hydrophilic petrolatum, possess hydrophilic properties that incorporate water to form w / o emulsions, yet maintain their semi-solid consistency. Finely divided solids are used as good emulsifiers, especially in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0279] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion, including fats, oils, waxes, fatty acids, fatty alcohols, fatty 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).

[0280] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers), which disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around droplets of the dispersed phase and by increasing the viscosity of the external phase.

[0281] Emulsions often contain many components, such as carbohydrates, proteins, sterols and phosphatides, which can easily support the growth of microorganisms, so these formulations often incorporate preservatives.The commonly used preservatives included in the formulation include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also usually added to emulsion formulations to prevent the formulation from deteriorating.The antioxidants used can be free radical scavengers, such as tocopherol, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents, such as ascorbic acid and sodium metabisulfite, and antioxidant synergists, such as citric acid, tartaric acid and lecithin.

[0282] The application of emulsion formulations via dermal, oral and parenteral routes as well as their manufacturing methods have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of formulation and effectiveness in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins and high fat nutritional formulations are among the materials commonly administered orally as o / w emulsions.

[0283] ii. Microemulsions In one embodiment of the present invention, the iRNA and nucleic acid compositions are formulated as microemulsions. Microemulsions can be defined as a system of water, oil and amphiphiles 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 oil in an aqueous surfactant solution, followed by the addition of a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a transparent system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared using a combination of three to five components including oil, water, surfactants, cosurfactants and electrolytes. Whether a microemulsion is of the water-in-oil (w / o) or oil-in-water (o / w) type depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0284] The phenomenological approach using phase diagrams has been extensively studied and provides those skilled in the art with extensive knowledge on how to formulate microemulsions (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that form spontaneously.

[0285] Surfactants used in the preparation of microemulsions, alone or in combination with cosurfactants, include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750). The cosurfactants, which are usually short chain alcohols such as ethanol, 1-propanol, and 1-butanol, serve to increase the interfacial fluidity by penetrating the surfactant film and thus forming an irregular film due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can include materials such as, but not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0286] Microemulsions are of particular interest from the standpoint of drug solubilization and drug absorption enhancement. Lipid-based microemulsions (both o / w and w / o) have been proposed to improve the oral bioavailability of drugs, including peptides (see, for example, U.S. Pat. Nos. 6,191,105, 7,063,860, 7,070,802, 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, protection of drugs from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Pat. Nos. 6,191,105, 7,063,860, 7,070,802, 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.Pharm.Sci., 1996, 85, 138-143). In many cases, microemulsions can form spontaneously when the components of the microemulsion are brought together at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or iRNAs. Microemulsions are also useful for the co-delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract, as well as improved local cellular uptake of iRNA and nucleic acids.

[0287] The microemulsions of the present invention may also include additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and permeation enhancers, to improve the properties of the formulation and enhance the absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories--surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0288] iii. Particulates The RNAi agents of the present invention may be incorporated into particles, such as microparticles. The 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.

[0289] iv. Penetration enhancers In one embodiment, the present invention uses various penetration enhancers to achieve efficient delivery of nucleic acids, particularly iRNA, to the skin of animals. Most drugs exist in solution in both ionized and non-ionized forms. However, usually only lipid-soluble or lipophilic drugs can easily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the permeability of lipophilic drugs.

[0290] Penetration enhancers may be classified as belonging to one of five broad categories, namely, surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above types of penetration enhancers is described in more detail below.

[0291] Surfactants (or "surface active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of the iRNA through mucosa. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether) (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorinated emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0292] Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1~20 Included are alkyl esters (e.g., methyl, isopropyl, and t-butyl), as well as their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0293] The physiological roles of bile include facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). A variety of natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharma- ceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee, J. Am. Chem. Soc. 1999, 144:1311-1323, 2002; Lee, J. Am. Chem. Soc. 1999, 144:1311-1323, 2002). et al.,Critical Reviews in Therapeutic Drug Carrier Systems,1991,page 92;Swinyard,Chapter 39 In:Remington's Pharmaceutical Sciences,18th Ed.,Gennaro,ed.,Mack Publishing Co.,Easton,Pa.,1990,pages 782-783;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0294] Chelators used in connection with the present invention may be defined as compounds that remove metal ions from solution by forming complexes with the metal ions, resulting in improved absorption of iRNA through mucous membranes. With regard to the use of chelators as penetration enhancers in the present invention, chelators have the added advantage of also acting as DNase inhibitors, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelators (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (see, e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0295] As used herein, non-chelating, non-surfactant penetration enhancers may be defined as compounds that exhibit little activity as chelators or surfactants, but nevertheless enhance the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0296] Agents that enhance the uptake of iRNA at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids such as lipofectin (Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., PCT Application WO 97 / 30731) are also known to enhance the cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), RNAiMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;(Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass; aD1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM) / LipoGen(TM)(Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent(Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent(Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent(Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER(TM) transfection Reagent (Genlantis; San Examples of suitable FECTOR vectors include RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA).

[0297] Other agents can be used to enhance the penetration of the administered nucleic acid, including glycols, such as ethylene glycol and propylene glycol, pyrroles, such as 2-pyrrole, azone, and terpenes, such as limonene and menthone.

[0298] v. Carrier Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analog that can be inert (i.e., does not possess biological activity) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading biologically active nucleic acids or facilitating the removal of nucleic acids from circulation. Co-administration of nucleic acids and carrier compounds, typically with an excess of the latter substance, can substantially reduce the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, possibly due to competition between the carrier compound and the nucleic acid for a common receptor. For example, recovery of partial phosphorothioate in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0299] vi. Excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid, and is selected to provide the desired bulk, consistency, etc., when combined with the nucleic acid and other predetermined components of the pharmaceutical composition, taking into account the planned method of administration. Typical pharmaceutical carriers include, but are not limited to, binders (such as, for example, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as, for example, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate); lubricants (such as, for example, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); tablet disintegrants (such as, for example, starch, sodium starch glycolate, and the like); and wetting agents (such as, for example, sodium lauryl sulfate, and the like).

[0300] Suitable organic or inorganic excipients that are pharma- ceutically acceptable for parenteral administration and do not adversely react with nucleic acids may also be used to formulate the compositions of the present invention. Suitable pharma-ceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0301] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions, or solutions of nucleic acids in liquid or solid oil bases in common solvents such as alcohol. The solutions can also contain buffers, diluents, and other suitable additives. Suitable organic or inorganic excipients that are pharma- ceutically acceptable for parenteral administration and do not adversely react with nucleic acids can be used.

[0302] Suitable pharma- ceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0303] vii. Other components The compositions of the present invention may also contain other auxiliary components conventionally found in pharmaceutical compositions, at their use levels established in the art. Thus, for example, the compositions may contain additional, compatible, pharma- ceutical active materials, such as, for example, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, these materials must not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized, and if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0304] The aqueous suspension may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain a stabilizer.

[0305] In some embodiments, the pharmaceutical compositions featured in the present invention include (a) one or more iRNA compounds and (b) one or more agents that function by non-RNAi mechanisms and are useful for treating lipid metabolism disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-steatotic agents, antiviral agents, and / or anti-fibrotic agents. In addition, other substances commonly used to protect the liver, such as silymarin, may also be used with the iRNAs described herein. Other agents useful for treating liver disease include protease inhibitors such as telbivudine, entecavir, and telaprevir, as well as other agents disclosed, for example, in Tung et al., U.S. Patent Application Publication Nos. 2005 / 0148548, 2004 / 0167116, and 2003 / 0144217; and Hale et al., U.S. Patent Application Publication No. 2004 / 0127488.

[0306] The toxicity and therapeutic efficacy of such compounds are described, for example, in LD 50 (lethal dose for 50% of the population) and ED 50 The LD (the dose therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Compounds that exhibit high therapeutic indices are preferred.

[0307] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosages of the compositions featured herein in the present invention are generally sufficient to achieve the ED with little or no toxicity. 50The dosage may vary within this range depending on the dosage form and route of administration used. For any compound used in the methods featured in the present invention, a therapeutically effective dose may be initially estimated from cell culture assays. The dose may be formulated to achieve in an animal model a circulating plasma concentration range of the compound or, where appropriate, the polypeptide product of the target sequence (e.g., achieve a reduced concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as measured in cell culture. Such information may be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0308] In addition to those administrations described above, the iRNAs featured in the present invention may be administered in combination with other known agents effective in treating pathological processes mediated by ANGPTL3 expression. In either case, the administering physician can adjust the amount and time of administration of the iRNA based on the observed results, using standard measures of efficacy known in the art or described herein.

[0309] VI. Methods of the Invention The present invention also provides a method of using the iRNA of the present invention and / or a composition containing the iRNA of the present invention to reduce and / or inhibit the expression of ANGPTL3 in a cell. The method includes contacting a cell with a dsRNA of the present invention and maintaining the cell for a sufficient time to obtain the degradation of the mRNA transcript of the ANGPTL3 gene, thereby inhibiting the expression of the ANGPTL3 gene in the cell. The reduction in gene expression can be evaluated by any method known in the art. For example, the reduction in the expression of ANGPTL3 can be measured by measuring the mRNA expression level of ANGPTL3 using methods that are routine for those skilled in the art, such as Northern blotting, qRT-PCR; by measuring the protein level of ANGPTL3 using methods that are routine for those skilled in the art, such as Western blotting, immunological techniques, etc. The reduction in the expression of ANGPTL3 can also be indirectly evaluated by measuring the reduction in the biological activity of ANGPTL3, such as the reduction in the levels of serum lipids, triglycerides, cholesterol and / or free fatty acids.

[0310] In the methods of the invention, the cells may be contacted in vitro or in vivo, i.e., the cells may be within a subject.

[0311] The cell suitable for treatment with the method of the present invention can be any cell that expresses ANGPTL3 gene.The cell suitable for use with the method of the present invention can be mammalian cell, for example primate cell (human cell or non-human primate cell, for example monkey cell or chimpanzee cell), non-primate cell (cow cell, pig cell, camel cell, llama cell, horse cell, goat cell, rabbit cell, sheep cell, hamster, guinea pig cell, cat cell, dog cell, rat cell, mouse cell, lion cell, tiger cell, bear cell or buffalo cell), bird cell (for example duck cell or goose cell), or whale cell.In one embodiment, the cell is human cell, for example human hepatocyte.

[0312] ANGPTL3 expression is at least about 5, 6, 7, 8, 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, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 109, 108, 109, 109, 101, 102, 103, 104, 105, 106, 107, 108, 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% inhibited.

[0313] The in vivo method of the present invention may include administering to a subject a composition containing an iRNA, the iRNA comprising a nucleotide sequence complementary to at least a portion of an RNA transcript of the ANGPTL3 gene of the mammal to be treated. When the organism to be treated is a mammal, such as a human, the composition may be administered by any means known in the art, including parenteral routes, including but not limited to oral, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

[0314] In some embodiments, administration is by depot injection. Depot injection can consistently release iRNA over a long period of time. Therefore, depot injection can reduce the frequency of administration required to obtain a desired effect, such as a desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. Depot injection can also provide a more consistent serum concentration. Depot injection can include subcutaneous injection or intramuscular injection. In a preferred embodiment, depot injection is a subcutaneous injection.

[0315] In some embodiments, administration is by pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In another embodiment, the pump is an infusion pump. The infusion pump can be used for intravenous, subcutaneous, intraarterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In another embodiment, the pump is a surgically implanted pump that delivers the iRNA to the liver.

[0316] The mode of administration may be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration may be selected to enhance targeting.

[0317] In one aspect, the present invention also provides a method for inhibiting the expression of ANGPTL3 gene in a mammal. The method includes administering a composition containing dsRNA targeting ANGPTL3 gene in a cell of the mammal to the mammal, and maintaining the mammal for a sufficient time to obtain the degradation of mRNA transcript of ANGPTL3 gene, thereby inhibiting the expression of ANGPTL3 gene in the cell. The reduction in gene expression can be evaluated by any method known in the art and described herein, for example, qRT-PCR. The reduction in protein production can be evaluated by any method known in the art and described herein, for example, ELISA. In one embodiment, a puncture liver biopsy sample serves as tissue material for monitoring the reduction in ANGPTL3 gene and / or protein expression.

[0318] The present invention further provides a method of treating a subject in need thereof, comprising administering a therapeutically effective amount of an iRNA of the present invention targeting the ANGPTL3 gene or a pharmaceutical composition comprising an iRNA targeting the ANGPTL3 gene to a subject, e.g., a subject who may benefit from reduced and / or inhibited expression of ANGPTL3.

[0319] The iRNA of the present invention may be administered as a "free iRNA". Free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer. The buffer may include acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS). The pH and osmolality of the buffer containing the iRNA may be adjusted as appropriate for administration to a subject.

[0320] Alternatively, the iRNA of the present invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

[0321] The subject that can benefit from the reduction and / or inhibition of ANGPTL3 gene expression is a subject suffering from lipid metabolism disorder, for example, a genetic lipid metabolism disorder or an acquired lipid metabolism disorder. In one embodiment, the subject suffering from lipid metabolism disorder suffers from hyperlipidemia. In another embodiment, the subject suffering from lipid metabolism disorder suffers from hypertriglyceridemia. The treatment of the subject that can benefit from the reduction and / or inhibition of ANGPTL3 gene expression includes therapeutic treatment (e.g., the subject suffers from eruptive xanthomas) and preventive treatment (e.g., the subject does not suffer from eruptive xanthomas or the subject may be at risk of developing eruptive xanthomas).

[0322] The present invention further provides a method for using iRNA or its pharmaceutical composition for treating subjects who may benefit from, for example, the reduction and / or inhibition of the expression of ANGPTL3, such as subjects suffering from lipid metabolism disorders, in combination with other pharmaceuticals and / or other treatment methods, such as, for example, known pharmaceuticals and / or known treatment methods, such as those currently used to treat these disorders. For example, in certain embodiments, iRNA targeting ANGPTL3 is administered in combination with an agent useful for treating lipid metabolism disorders, such as, for example, those described elsewhere herein. For example, additional agents suitable for treating subjects who may benefit from the reduction of the expression of ANGPTL3, such as subjects suffering from lipid metabolism disorders, may include one or more serum lipid lowering agents. Non-limiting examples of such agents may include cholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors, for example, statins. Statins may include atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor), sustained release lovastatin (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin (Zocor).Other agents useful for treating lipid metabolism disorders may include bile sequestering agents such as cholestyramine and other resins; VLDL secretion inhibitors such as niacin; lipophilic antioxidants such as probucol; acyl-CoA cholesterol acyltransferase inhibitors; farnesoid X receptor antagonists; sterol regulatory binding protein cleavage activating protein (SCAP) activators; microsomal triglyceride transfer protein (MTP) inhibitors; ApoE-related peptides; and therapeutic antibodies against ANGPTL3. The additional therapeutic agents may also include agents that increase high density lipoprotein (HDL), such as cholesteryl ester transfer protein (CETP) inhibitors. Additionally, the additional therapeutic agents may also include nutritional supplements, such as fish oil.The iRNA and additional therapeutic agent may be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agent may be administered as part of a separate composition, or at different times, and / or by other methods known in the art or described herein.

[0323] In one embodiment, the method includes administering a composition featured herein such that expression of the target ANGPTL3 gene is reduced for, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, or about 36 hours. In one embodiment, expression of the target ANGPTL3 gene is reduced for an extended duration, e.g., at least about 2, 3, 4 days or more, e.g., about 1 week, 2 weeks, 3 weeks, or 4 weeks or more.

[0324] Preferably, the iRNAs useful in the methods and compositions featured herein specifically target the RNA (primary RNA or processed RNA) of the target ANGPTL3 gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

[0325] The administration of dsRNA according to the method of the present invention can reduce the severity, signs, symptoms, and / or markers of such disease or disorder in patients with lipid metabolism disorders.In this context, "reduction" refers to a statistically significant reduction in their levels.Reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0326] The effectiveness of treatment or prevention of disease can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of drug required to sustain the treatment effect, the level of disease markers, or any other measurable parameter appropriate for the given disease being treated or targeted for prevention. It is well within the skill of the skilled artisan to monitor the effectiveness of treatment or prevention by measuring any one of these parameters or any combination of parameters. For example, the effectiveness of treatment of lipid metabolism disorders can be evaluated, for example, by periodic monitoring of one or more serum lipid levels. Comparison of early and later readings gives the physician an indication of whether the treatment is effective. It is well within the skill of the skilled artisan to monitor the effectiveness of treatment or prevention by measuring any one of these variables or any combination of variables. In connection with administration of an iRNA targeting ANGPTL3 or a pharmaceutical composition thereof, "effective against" a lipid metabolism disorder indicates that administration in a clinically relevant manner will result in a beneficial effect in at least a statistically significant percentage of patients, such as amelioration of symptoms, cure, reduction of disease, extension of life span, improvement in quality of life, or other effect generally recognized as favorable by physicians knowledgeable in the treatment of lipid metabolism disorders and related causes.

[0327] The efficacy of treatment or prevention is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when a worsening or otherwise expected symptom does not occur. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more, in a measurable parameter of the disease may indicate an effective treatment. The efficacy of an iRNA drug, or a given formulation of the drug, may also be determined using an experimental animal model for a given disease, as known in the art. When using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant decrease in a marker or symptom is observed.

[0328] Alternatively, efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity rating scale, such as, for example, the Child-Pugh score (sometimes referred to as the Child-Turcotte-Pugh score). Any positive change, e.g., resulting in a reduction in disease severity, measured using an appropriate scale, is indicative of sufficient treatment with an iRNA or iRNA formulation described herein.

[0329] The subject is administered about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 5 mg / kg, Approximately 0.3mg / kg to approximately 10mg / kg, approximately 0.4mg / kg to approximately 5mg / kg, approximately 0.4mg / kg to approximately 10mg / kg, approximately 0.5mg / kg to approximately 5mg / kg, approximately 0.5mg / kg to approximately 10 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 2 mg / kg to about 2. 5mg / kg, about 2mg / kg to about 10mg / kg, about 3mg / kg to about 5mg / kg, about 3mg / kg to about 10mg / kg, about 3.5mg / kg to about 5mg / kg, about 4mg / kg to about 5m g / kg, about 4.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5.5 mg / kg to about A therapeutic amount of dsRNA may be administered, such as 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​up to and including the cited values ​​and intermediate ranges are contemplated as part of the present invention.

[0330] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or about 10 mg / kg. Values ​​up to and including the recited values ​​and intermediate ranges are contemplated as part of the invention.

[0331] In another embodiment, for example, when the composition of the present invention contains a dsRNA and N-acetylgalactosamine described herein, the subject is administered about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg , about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 5 0mg / kg, about 0.1 to about 45mg / kg, about 0.25 to about 45mg / kg, about 0.5 to about 45mg / kg, about 0.75 to about 45mg / kg, about 1 to about 45mg / mg, about 1.5 to about 45mg / kb, about 2 to about 45mg / kg, about 2.5 to about 45mg / kg, about 3 to about 45m g / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 ~40mg / mg, 1.5~40mg / kb, 2~40mg / kg, 2.5~40mg / kg, 3~40mg / kg, 3.5~40mg / kg, 4~40mg / kg, 4.5~40mg / kg, 5~40mg / kg, 7.5~40mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg , about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about Therapeutic amounts of dsRNA may be administered, such as doses of 0.1 to about 20 mg / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and intermediate ranges up to the cited values ​​are intended to be part of the invention.

[0332] For example, the subject may be administered about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5 ,7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 1 Therapeutic amounts of dsRNA such as 7, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg may be administered. Values ​​up to and including the cited values ​​and intermediate ranges are contemplated as part of the invention.

[0333] The iRNA may be administered by intravenous infusion over a period of time, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 minutes. Administration may be repeated, for example, periodically, for example, every other week (i.e., every two weeks) for one month, two months, three months, four months, or longer. After an initial treatment regimen, treatment may be administered less frequently. For example, after three months of administration every other week, administration may be repeated once a month for six months, or one year, or longer. Administration of an iRNA can, for example, increase ANGPTL3 levels in cells, tissues, blood, urine, or other compartments of a patient by at least about 5%, , 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more.

[0334] Prior to administering the full dose of iRNA, the patient may be administered a smaller dose (e.g., a 5% infusion reaction) and monitored for adverse effects such as allergic reactions. In another example, the patient may be observed for unwanted immunostimulatory effects such as increased cytokine (e.g., TNF-α or INF-α) levels.

[0335] Alternatively, the iRNA may be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to the subject. The injections may be repeated for a predetermined period of time, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 days. Administration may be repeated periodically, such as, for example, every other week (i.e., every two weeks) for one month, two months, three months, four months, or more. After an initial treatment regimen, the treatment may be administered less frequently. In some embodiments, a single dose of iRNA is followed by monthly dosing. In some embodiments, administration may include a loading phase of multiple doses on consecutive days.

[0336] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Similar or equivalent methods and materials as described herein can be used to practice or test the iRNA and methods that characterize the present invention, and suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting. EXAMPLES

[0337] Example 1 iRNA synthesis Reagent Sources If the source of a reagent is not specifically given herein, the reagents may be obtained from any supplier in molecular biology and of quality / purity standards for use in molecular biology.

[0338] Transcripts siRNA design was performed to identify siRNAs targeting human ANGPTL3 transcripts annotated in the NCBI Gene database (http: / / www.ncbi.nlm.nih.gov / gene / ) and cynomolgus monkey (Macaca fascicularis; hereafter "cyno") ANGPTL3 transcripts generated by sequencing cDNA prepared from liver RNA. Sequencing of cyno ANGPTL3 mRNA was performed in-house. The mRNA sequence is shown in SEQ ID NO:9.

[0339] The following transcripts from the NCBI collection were used for the design: human-NM_014495.2 (SEQ ID NO: 1); mouse-NM_013913.3 (SEQ ID NO: 2). All siRNA duplexes were designed that shared 100% identity with the listed human and cyno transcripts. A subset of siRNA duplexes, described below, also shared 100% identity with mouse (Mus musculus) ANGPTL3 transcripts found in the NCBI Gene database.

[0340] siRNA design, specificity, and efficacy prediction The predicted specificity of all possible 19-mers was predicted from each sequence. Candidate 19-mers with no repeats longer than 7 nucleotides were then selected. These 977 candidate human / cyno siRNAs, and a subset of 38 that also matched mouse ("human / cyno / mouse candidate siRNAs"), were then used in a comprehensive search against the human transcriptome (defined as the set of NM_ and XM_ records in the human NCBI Refseq set) using a comprehensive "brute-force" algorithm implemented in the python script "BruteForce.py". The script then analyzes the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any possible "off-target" transcripts. The off-target score is weighted to highlight differences in the "seed" region of the siRNA, located 2-9 positions from the 5' end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; a mismatch at positions 2-9 was considered 2.8, a mismatch at cleavage site 10-11 was considered 1.2, and a mismatch in region 12-19 was considered 1.0. Further off-target prediction was performed by comparing the frequency of heptamers and octamers derived from three different, seed-directed hexamers for each oligo. Two heptamers and one octamers were generated using hexamers from positions 2-7 relative to the 5' start. "Heptamer 1" was generated by adding a 3'A to the hexamer; "Heptamer 2" was generated by adding a 5'A to the hexamer; and an octamers was generated by adding an A to both the 5' and 3' ends of the hexamer. We previously calculated the frequencies of 8-mers and 7-mers in the human 3'UTRome (defined as a subsequence of the transcriptome from NCBI's Refseq database with clearly defined ends of coding sequences (CDSs)). The median value from the range of 8-mer frequencies was used to normalize the 8-mer frequencies to the 7-mer frequencies.The “mirSeedScore” was then calculated by calculating the sum of ((3 × normalized 8mer value) + (2 × heptamer2 value) + (1 × heptamer1 value)).

[0341] Both siRNA strands were assigned to specificity categories according to the calculated score: a score above 3 is considered highly specific, a score equal to 3 is considered specific, and a score between 2.2 and 2.8 is considered moderately specific. Sorting was performed according to the specificity of the antisense strand. Next, duplexes were selected from the human / cyno set that had antisense oligos with no miRNA seed match (score ≥ 3, total GC content < 65%, no GC at the 1st position, ≥ 4 Us or As in the seed region, and GC at the 19th position). Also, duplexes were selected from the human / cyno / mouse set that had antisense oligos with score ≥ 2, total GC content < 65%, and no GC at the 1st position.

[0342] Selection of siRNA sequences A total of 47 sense and 47 antisense siRNA oligos derived from the human / cyno set were synthesized and duplexed. A total of 15 sense and 15 antisense siRNA oligos derived from the human / cyno / mouse set were synthesized and duplexed.

[0343] Synthesis of ANGPTL3 sequence ANGPTL3 sequences were synthesized on a MerMade 192 synthesizer at either 1 or 0.2 μmol scale. Single strands with 2′O-methyl modifications were synthesized for transfection-based in vitro screening. 3′GalNAc conjugates with 2′F and 2′-O-methyl chemical modifications were generated for use in free-uptake screening assays. In these designs, the GalNAc moiety was placed at the 3′ end of the sense strand. The antisense sequences were 23 nucleotides long and contained 2′F and 2′O-methyl chemical modifications with two phosphorothioate bonds at the 3′ end.

[0344] On a set of 21-mer single and double strands, "endolight" chemistry was applied, as detailed below. · All pyrimidines (cytosines and uridines) in the sense strand were modified with 2'-O-methyl nucleotides (2'-O-methyl C and 2'-O-methyl U). In the antisense strand, pyrimidines adjacent (towards the 5' position) to ribo A nucleosides were replaced with their corresponding 2'-O-methyl nucleosides. A two base dTsdT extension was introduced at the 3' end of both the sense and antisense sequences.

[0345] For GalNAc-conjugated 21-mer sense and complementary 23-mer antisense sequences, 2'F and 2'O methyl modified single strands were synthesized. Synthesis was performed on a 1 μmol scale on GalNAc-modified CPG support for the sense strand and on CPG modified with universal support for the antisense sequence. A sequence motif named TOFFEE was applied, where the sense strand contained a 3-nucleotide 2'F modified motif at positions 9, 10 and 11, and in the antisense, a 2'O methyl modified motif was contained at positions 11, 12 and 13.

[0346] Synthesis, cleavage and deprotection Synthesis of ANGPTL3 sequences used solid-supported oligonucleotide synthesis using phosphoramidite chemistry. For the 21-mer endlite sequences, deoxythymidine CPG was used as the solid support, while GalNAc conjugates used GalNAc solid support for the sense strand and universal CPG for the antisense strand.

[0347] The synthesis of the above sequences was carried out at either 1 or 0.2 μm scale in 96-well plates. Amidite solutions were prepared at a concentration of 0.1 M and ethylthiotetrazole (0.6 M in acetonitrile) was used as the activating agent.

[0348] The synthesized sequences were cleaved and deprotected in 96-well plates using methylamine in the first step and fluoride reagent in the second step. For sequences containing GalNAc and 2'F nucleosides, the deprotection conditions were modified. Sequences after cleavage and deprotection were precipitated using an acetone:ethanol (80:20) mixture and the pellet was resuspended in 0.2 M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm identity, UV for quantification, and a selected set of samples by IEX chromatography to measure purity.

[0349] Purification, desalting and annealing The ANGPTL3 sequence was precipitated and purified on an AKTA Purifier system using a Sephadex column. ANGPTL3 was run at ambient temperature. Sample injection and collection were performed in a 96-well plate with wells 1.8 mL deep. A single peak corresponding to the full-length sequence was collected in the eluate. The desalted ANGPTL3 sequence was purified at a concentration (A 260 The complementary single strands were then combined in a 1:1 stoichiometric ratio to form the siRNA duplex.

[0350] Example 2. In vitro screening Cell culture and transfection Hep3B cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) in an atmosphere of 5% CO2 and then released from the plate by trypsinization. Transfection was performed by adding 14.8 μl Opti-MEM + 0.2 μl Lipofectamine RNAiMax (Invitrogen, Carlsbad CA. cat#13778-150 per well to 5 μl siRNA duplexes per well in a 96-well plate and incubated at room temperature for 15 minutes. Then, ∼2 × 10 4 80 μl of complete growth medium containing Hep3B cells was added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Unless otherwise indicated, single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations, and dose response experiments were performed at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM final duplex concentrations.

[0351] Free uptake transfection 5 μl of each GalNac-conjugated siRNA in PBS was added to each well of a 96-well plate, along with 4 × 10 siRNA resuspended in 95 μl of In Vitro Gro CP medium (In Vitro Technologies-Celsis, Baltimore, MD). 4 The siRNA was combined with freshly thawed cryopreserved cynomolgus monkey hepatocytes. The mixture was incubated at 37° C. in a 5% CO2 atmosphere for approximately 24 hours. For efficacy free uptake assays, siRNA was tested at final concentrations of 500 nM, 100 nM, and 10 nM. For dose-response screening, the final siRNA concentrations were 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.0064 nM.

[0352] Total RNA isolation using DYNABEADS mRNA isolation kit (Invitrogen, part#:610-12): The cells were harvested and lysed in 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (mixing speed was the same throughout the process). 10 microliters of magnetic particles and 80 μl of lysis / binding buffer mixture were added to the round bottom plate and mixed for 1 minute. The magnetic particles were captured using a magnetic stand and the supernatant was removed without disturbing the particles. After removal of the supernatant, the lysed cells were added to the remaining particles and mixed for 5 minutes. After removal of the supernatant, the magnetic particles were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The particles were captured again and the supernatant was removed. The particles were then washed with 150 μl of wash buffer B, captured and the supernatant was removed. The particles were then washed with 150 μl of elution buffer, captured and the supernatant was removed. The particles were dried for 2 minutes. After drying, 50 μl of elution buffer was added and mixed for 5 minutes at 70° C. The particles were captured on the magnet for 5 minutes. 40 μl of the supernatant was removed and added to another 96-well plate.

[0353] cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat# 4368813): A master mix consisting of 2 μl 10X buffer, 0.8 μl 25X dNTPs, 2 μl random primers, 1 μl reverse transcriptase, 1 μl RNAe inhibitor, and 3.2 μl H2O was added to 10 μl total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermocycler (Hercules, CA) through the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 sec, hold at 4°C.

[0354] Real-time PCR: 2 μl of cDNA was added to a master mix containing 0.5 μl of GAPDH TaqMan probe (Applied Biosystems Cat#4326317E), 0.5 μl of ANGPTL3 TaqMan probe (Applied Biosystems cat#Hs00163644_m1) and 5 μl of Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384-well 50 plate (Roche cat#04887301001). Real-time PCR was performed in an ABI7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt (RQ) assay. Unless otherwise noted in the schematic table, each duplex was tested in two independent transfections and each transfection was assayed in duplicate.

[0355] To calculate relative fold changes, real-time data was analyzed using the ΔΔCt method and normalized to assays performed with 10 nM AD-1955-transfected cells or mock-transfected cells. IC50s were calculated using a four-parameter fit model with XLFit and normalized to AD-1955-transfected cells or untreated cells over the same dose range or normalized to the lowest dose itself. The AD-1955 sequence used as a negative control targeted luciferase and had the following sequence: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14); antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 15).

[0356] Viability Screening Cell viability was measured in HeLa and Hep3B cells on days 3 and 6 after transfection with 10, 1, 0.5, 0.1, 0.05 nM siRNA. Cells were plated at a density of 10,000 cells per well of a 96-well plate. Each siRNA was assayed in triplicate and data were averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for reduced viability, and AD-1955 and mock-transfected cells were included as negative controls. PLK1 and AD-19200 resulted in a dose-dependent reduction in viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of the 96-well plate after 3 or 6 days and incubated at 37° C. for 2 hours. Plates were then read in a spectrophotometer (Molecular Devices) at 560Ex / 590Em. Viability was expressed as the average of light units from three replicate transfections + / - the standard deviation. Relative viability was assessed by first averaging the three replicate transfections and then normalizing to mock-transfected cells. Data are expressed as % of viable cells.

[0357] Table 1: Abbreviations for nucleotide monomers used in the description of nucleic acid sequences. It will be understood that these monomers, when present in an oligonucleotide, are joined to each other by 5'-3'-phosphodiester bonds.

[0358] [Table 4]

[0359] [Table 5]

[0360] [Table 6]

[0361] [Table 7]

[0362] [Table 8]

[0363] [Table 9]

[0364] [Table 10]

[0365] [Table 11]

[0366] [Table 12]

[0367] Table 4. Results of single-dose screening using ANGPTL3 dsRNA sequences Experiments were performed with the modified oligonucleotide duplexes listed in Table 3. The sequence of AD-15838.2 is identical to that of AD-15838.1. Delivery of the siRNA duplexes was performed using LNPs.

[0368] [Table 13]

[0369] [Table 14]

[0370] [Table 15]

[0371] Table 5. Dose-response screening results for ANGPTL3 dsRNA sequences Experiments were performed with the modified oligonucleotide duplexes listed in Table 3. The sequence of AD-15838.2 is identical to that of AD-15838.1.

[0372] [Table 16]

[0373] Table 6. Results of cell viability screening using modified ANGPTL3 dsRNA sequences Experiments were performed using the modified oligonucleotide duplexes listed in Table 3. The sequence of AD-15838.2 is identical to that of AD-15838.1. Viability data are expressed as % viability compared to mock treated cells.

[0374] [Table 17]

[0375] [Table 18]

[0376] [Table 19]

[0377] [Table 20]

[0378] [Table 21]

[0379]

Table 22

[0380]

Table 23

[0381]

Table 24

[0382]

Table 25

[0383]

Table 26

[0384]

Table 27

[0385]

Table 28

[0386]

Table 29

[0387]

Table 30

[0388]

Table 31

[0389]

Table 32

[0390] [Table 33]

[0391] [Table 34]

[0392] [Table 35]

[0393] [Table 36]

[0394] [Table 37]

[0395] [Table 38]

[0396] [Table 39]

[0397] [Table 40]

[0398] [Table 41]

[0399] Table 9. Unmodified sense and antisense strand sequences of ANGPTL3 dsRNA without GalNal conjugates These sequences are the same as those listed in Table 7, except that they do not contain the GalNal conjugate.

[0400] [Table 42]

[0401] [Table 43]

[0402] [Table 44]

[0403] [Table 45]

[0404] [Table 46]

[0405] [Table 47]

[0406] [Table 48]

[0407] [Table 49]

[0408] [Table 50]

[0409] [Table 51]

[0410] Table 10. Modified sense and antisense strand sequences of ANGPTL3 dsRNA without GalNal conjugates These sequences are the same as those listed in Table 8, except that they do not contain the GalNal conjugate.

[0411] [Table 52]

[0412] [Table 53]

[0413] [Table 54]

[0414] [Table 55]

[0415] [Table 56]

[0416] [Table 57]

[0417] [Table 58]

[0418] [Table 59]

[0419] [Table 60]

[0420] Table 11. Results of single dose screening with ANGPTL3 GalNac conjugated dsRNA The modified siRNAs were tested by transfection in Hep3b cells and by free uptake in primary cynomolgus monkey (PCH) cells at the doses mentioned above.

[0421] [Table 61]

[0422] [Table 62]

[0423] [Table 63]

[0424] [Table 64]

[0425] [Table 65]

[0426] [Table 66]

[0427] [Table 67]

[0428] [Table 68]

[0429] [Table 69]

[0430] [Table 70]

[0431] Table 12. Dose-response screening results for ANGPTL3 GalNac-conjugated dsRNA sequences. A subset of active siRNAs from the single dose screen (see data in Table 11) were tested in dose-response experiments by free uptake in PCH cells. A subset of these active siRNAs were also tested in dose-response in Hep3B cells by transfection.

[0432] [Table 71]

[0433] [Table 72]

[0434] [Table 73]

[0435] [Table 74]

[0436] [Table 75]

[0437] [Table 76]

[0438] [Table 77]

[0439] [Table 78]

[0440] Table 14. Results of dose-response screening using a subset of sequences from Table 13 A subset of active ANGPTL3 siRNAs from Table 10 were tested by transfection in Hep3B cells in a dose-response screen.

[0441] [Table 79]

[0442] [Table 80]

[0443] Table 15. IDs of duplex pairs synthesized and tested in both unconjugated and GalNac conjugated forms. These duplexes have the same sequence and modification pattern.

[0444] [Table 81]

[0445] [Table 82]

[0446] [Table 83]

[0447] [Table 84]

[0448] [Table 85]

[0449] [Table 86]

[0450] In vivo testing Example 3 Test substance In vivo experiments were performed using dsRNA sequences of the invention. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 ("ANG", sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842). The dsRNA sequence used as a negative control was luciferase-conjugated The target was AD-48399B1 ("Luc", sense sequence: CfaCfuUfaCfgCfuGfaGfuAfcUfuCfgAfL96 (SEQ ID NO: 1728), antisense sequence: uCfgAfaGfuAfcUfcAfgCfgUfaAfgUfgsAfsu (SEQ ID NO: 1729)). The GalNal conjugate AD-1955, which contains alternating 2'-methyl and 2' fluoro modifications, was also used as a negative control.

[0451] Experimental procedure dsRNA sequences were tested in C57BL / 6 (wild type (WT)) and ob / ob mice. Wild type (WT) mice were administered dsRNA in PBS, 20 mg / kg Luc, or 5 or 20 mg / kg ANG once a day for 5 days; ob / ob mice were administered 20 mg / kg NPL formulated with Luc or 20 mg / kg ANG once a day for 5 days. All test substances were administered by subcutaneous injection according to the procedure shown in Figure 1. Specifically, test substances were administered once a day for 5 days for 5 consecutive days (days 0, 1, 2, 3, and 4), and blood samples were taken 5, 3, or 1 day before administration and 0, 1, 2, 3, 4, 7, 9, 11, 15, 18, 21, 25, 30, 37, 45, and 50 days after administration. The collected blood samples were used to measure ANGPTL3 protein expression using an ELISA assay, and serum triglyceride (TG), low-density lipoprotein cholesterol (LDLc), high-density lipoprotein cholesterol (HDLc), and total cholesterol (TC) levels were measured using an Olympus Analyzer.

[0452] result Panel A shown in FIG. 2 is the level of mouse ANGPTL3 (mANGPTL3 protein) measured in wild-type (WT) mice after administration of 5 or 20 mg / kg of control or ANG. Panel B shown in FIG. 2 is the level of mANGPTL3 protein measured in ob / ob mice after administration of 20 mg / kg of control or ANG. The data show that for both wild-type (WT) and ob / ob mice, administration of ANG reduced the level of mANGPTL3 protein compared to controls.

[0453] Panel A shown in Figure 3 is the level of LDL-c measured in wild-type (WT) mice after administration of 20 mg / kg of control or ANG. Panel B shown in Figure 3 is the level of LDL-c measured in ob / ob mice after administration of 20 mg / kg of control or ANG. The data show that administration of ANG reduced the level of LDL-c, particularly in ob / ob mice, compared to controls.

[0454] Panel A shown in Figure 4 is the triglyceride levels measured in wild-type (WT) mice after administration of 20 mg / kg of control or ANG. Panel B shown in Figure 4 is the triglyceride levels measured in ob / ob mice after administration of 20 mg / kg of control or ANG. The data show that administration of ANG reduced triglyceride levels, particularly in ob / ob mice, compared to controls.

[0455] Panels A and B shown in Figure 5 are total cholesterol (TC) levels measured in wild-type (WT) and ob / ob mice, respectively, after administration of 20 mg / kg of control or ANG. The data show that administration of ANG moderately reduces TC levels in ob / ob mice, but not in wild-type (WT) mice. Similarly, as shown in the graph in Figure 6, administration of ANG moderately reduces HDL-c levels in ob / ob mice, but not in wild-type (WT) mice.

[0456] Example 4 Test substance The effect of a single administration of the dsRNA sequence of the present invention on ANGPTL3 protein level was tested.The dsRNA sequence used in the experiment was GalNac conjugate AD-52981 ("ANG", sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)).PBS was used as a negative control.

[0457] Experimental procedure The dsRNA sequences were tested in human PCS transgenic mice, which are characterized by liver-specific expression of the full-length human PCSK9 gene. Human PCS transgenic mice were administered AD-52981 or PBS using a single subcutaneous injection. Mice were divided into four groups, each group consisting of two males and two females. Each group was injected with PBS or administered 5 mg / kg, 20 mg / kg, or 60 mg / kg of AD-52981. Blood samples were taken 1 day and 0 day before administration, and 72 hours after administration. ANGPTL3 protein levels were measured by ELISA and compared to levels 1 day and 0 day before administration.

[0458] result Shown in Figure 7 are levels of mouse ANGPTL3 protein (mANGPTL3) measured in human PCS transgenic mice. Data shown are expressed relative to PBS controls and represent the average of two males and two females in each group. Error bars represent standard deviation. The data show that administration of a single injection of AD-52981 dose-dependently reduces ANGPTL3 protein levels in mice, with a dose of 60 mg / kg reducing ANGPTL3 protein levels by more than 5-fold (see Figure 7).

[0459] array SEQ ID NO:1 >gi|41327750|ref|NM_014495.2|Human (Homo sapiens) angiopoietin-like 3 (ANGPTL3), mRNA

[0460] SEQ ID NO:2 >gi|297278846|ref|XM_001086114.2|PREDICTED: Rhesus monkey (Macaca mulatta) angiopoietin-like 3 (ANGPTL3), mRNA

[0461] SEQ ID NO:3 >gi|142388354|ref|NM_013913.3|Mus musculus angiopoietin-like 3 (Angptl3), mRNA

[0462] SEQ ID NO:4 >gi|68163568|ref|NM_001025065.1|Rattus norvegicus angiopoietin-like 3 (Angptl3), mRNA

[0463] SEQ ID NO:5 Reverse complementary sequence of SEQ ID NO:1

[0464] SEQ ID NO:6 Reverse complementary sequence of SEQ ID NO:2

[0465] SEQ ID NO: 7 Reverse complementary sequence of SEQ ID NO: 3

[0466] SEQ ID NO:8 Reverse complementary sequence of SEQ ID NO:4

[0467] SEQ ID NO:9 Macaca fascicularis angiopoietin-like 3 (Angptl3), mRNA

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of angiopoietin-like 3 (ANGPTL3), comprising: the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length; the sense strand comprises at least 18 contiguous nucleotides from the nucleotide sequence 5'-AUUAAGCUCCUUCUUUUUAUU-3' (SEQ ID NO:283); the antisense strand comprises at least 18 contiguous nucleotides from the nucleotide sequence 5'-AAUAAAAAAGAAGGAGCUUAAUUG-3' (SEQ ID NO:468); every nucleotide of the sense strand and every nucleotide of the antisense strand comprises a nucleotide modification selected from the group consisting of a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, and a 2'-deoxy-nucleotide modification; and a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to the sense strand; dsRNA agents.

2. 10. The dsRNA agent of claim 1, wherein said ligand is conjugated to the 3' end of a sense strand of said dsRNA agent.

3. The ligand is 【Chemistry 1】 The dsRNA agent of claim 1 or 2,

4. The ligand is: 【Chemistry 2】 4. The dsRNA agent of claim 1, wherein the dsRNA agent is conjugated to the dsRNA agent as shown in

5. The dsRNA agent of any one of claims 1 to 4, further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

6. The dsRNA agent of claim 5, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand.

7. The dsRNA agent of claim 5, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand.

8. The dsRNA agent of claim 5, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of a single strand.

9. A cell comprising the dsRNA agent of any one of claims 1 to 8.

10. A pharmaceutical composition for inhibiting expression of the ANGPTL3 gene, comprising the dsRNA agent of any one of claims 1 to 8.

11. The pharmaceutical composition of claim 10, wherein the dsRNA agent is in a buffer solution.

12. The pharmaceutical composition of claim 10, wherein the dsRNA agent is in a non-buffered solution.

13. 1. An in vitro method of inhibiting expression of ANGPTL3 in a cell, comprising: a) contacting said cell with a dsRNA agent according to any one of claims 1 to 8; and b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cells. The method includes:

14. A pharmaceutical composition for treating a subject suffering from a disorder that may benefit from reduced expression of ANGPTL3, comprising the dsRNA agent of any one of claims 1-8.

15. The pharmaceutical composition of claim 14, wherein the disorder is a lipid metabolism disorder.

16. 16. The pharmaceutical composition of claim 14 or 15, wherein the disorder is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, atherosclerosis, diabetes, cardiovascular disease, and coronary artery disease.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein the disorder is hyperlipidemia.

18. The pharmaceutical composition of any one of claims 14 to 17, further comprising an additional therapeutic agent.

19. 20. The pharmaceutical composition of claim 18, wherein the additional therapeutic agent is a statin.

20. The pharmaceutical composition of any one of claims 14 to 19, wherein administration of the dsRNA agent to a subject causes a reduction in one or more serum lipids.

21. The pharmaceutical composition of any one of claims 14 to 20, wherein administration of the dsRNA agent to a subject causes a decrease in ANGPTL3 protein accumulation.

22. The pharmaceutical composition according to any one of claims 14 to 21, wherein the subject is a human subject.

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