Angiopoietin-like 3 (ANGPTL3) iRNA composition and method of use thereof

Abstract

To provide iRNA compositions which effect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPL3 gene.SOLUTION: A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3 comprises a sense strand and an antisense strand. The sense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from the nucleotide sequence of SEQ ID NO:1. The antisense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from the nucleotide sequence of SEQ ID NO:5.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted factors that regulate lipid metabolism and are mainly expressed in the liver (Non-Patent Document 1). ANGPTL3 doubly inhibits the catalytic activity of lipoprotein lipase (LPL), which hydrolyzes triglycerides and catalyzes the hydrolysis of endothelial lipase (EL), which hydrolyzes high-density lipoprotein (HDL) phospholipids. In KK / Snk mice that are still obese but have reduced lipids, the reduction in ANGPTL3 expression has a preventive effect against hyperlipidemia and atherosclerosis by promoting the clearance of triglycerides (Non-Patent Document 2). Human ANGPTL3 plasma concentration is 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 pathological conditions. Hypertriglyceridemia is an example of a lipid metabolism disorder characterized by high blood concentrations of triglycerides. Hypertriglyceridemia has been associated with atherosclerosis even in the absence of high cholesterol levels (hypercholesterolemia). When triglyceride concentrations are excessive (i.e., above 1000 mg / dl or 12 mmol / l), hypertriglyceridemia can also cause pancreatitis. Hyperlipidemia is another example of a lipid metabolism disorder characterized by an increase in the level of either or all of the lipids and / or lipoproteins in the blood. Current treatments for lipid metabolism disorders, including dietary therapy, exercise, and treatment with statins and other drugs, are not always effective. Therefore, alternative treatments for subjects suffering from lipid metabolism disorders are needed in the art.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0005] The present invention provides an iRNA composition that induces RNA-induced silencing complex (RISC) cleavage of the RNA transcript of the ANGPL3 gene. The ANGPL3 gene may be present in cells, for example, in cells in subjects such as humans. The present invention also provides methods for using the iRNA composition of the present invention to inhibit the expression of the ANGPL3 gene and / or to treat subjects who may benefit from inhibiting or reducing the expression of the ANGPL3 gene, such as subjects who have or are susceptible to lipid metabolic disorders, such as subjects who have or are susceptible to hyperlipidemia or hypertriglyceridemia.

[0006] Accordingly, in one embodiment, 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 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 5 by three or fewer nucleotides.

[0007] In another embodiment, 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, the antisense strand comprising a complementary region containing at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in Tables 2, 3, 7, 8, 9, and 10 by three or fewer nucleotides.

[0008] In one embodiment, the sense strand and antisense strand include sequences 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, and AD-53077.1 as shown 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 containing a 5'-phosphorothioate group, and terminal nucleotides bonded to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. 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, nonbasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing non-natural bases.

[0010] The complementary region of dsRNA may be at least 17 nucleotides long, 19-21 nucleotides long, or 19 nucleotides long.

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

[0012] At least one strand of dsRNA may contain a 3' overhang on 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 bound to the 3' end of the sense strand of the dsRNA.

[0014] In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives linked via a divalent or trivalent branched linker. In a particular embodiment, the ligand is [ka] That is the case.

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

[0016] In one embodiment, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond. In one embodiment, the phosphorothioate or methylphosphonate internucleotide bond is located at the 3' end of a single strand. In one embodiment, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

[0017] In one embodiment, the complementary region of the dsRNA consists of one of the antisense sequences shown 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 in Tables 2, 3, 7, 8, 9, and 10, and an antisense strand consisting of an antisense sequence selected from the sequences in Tables 2, 3, 7, 8, 9, and 10.

[0019] In another embodiment, the present invention provides cells containing the dsRNA of the present invention, for example, hepatocytes.

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

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

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

[0023] In one embodiment, the pharmaceutical composition includes a lipid preparation such as MC3, SNALP, or XTC preparation.

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

[0025] The cells may be present in human subjects, such as human subjects suffering from lipid metabolism disorders, such as hyperlipidemia or hypertriglyceridemia.

[0026] In one embodiment of the method of the present invention, ANGPTL3 expression 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 embodiment, the present invention provides a method for treating subjects suffering from lipid metabolism disorders such as hyperlipidemia or hypertriglyceridemia, which may benefit from reduced ANGPTL3 expression. The method comprises administering a therapeutically effective amount of the present invention's dsRNA or vector to a subject, thereby treating the subject.

[0028] The injury may be a lipid metabolism disorder such as hyperlipidemia or hypertriglyceridemia.

[0029] In one embodiment, administration of dsRNA to a subject causes a decrease in serum lipid, triglyceride, cholesterol and / or free fatty acid levels; and / or a decrease in ANGPTL3 protein accumulation. In one embodiment, administration of dsRNA to a subject causes a decrease in LDL-C, HDL-C, VLDL-C, IDL-C and / or total cholesterol levels.

[0030] In one embodiment, the dsRNA concentration is approximately 0.01 mg / kg to approximately 10 mg / kg, for example, approximately 0.05 mg / kg to approximately 5 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 0.2 mg / kg to approximately 5 mg / kg, approximately 0.2 mg / kg to approximately 10 mg / kg, and approximately 0.3 mg / kg to approximately 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. It is administered in doses of approximately 5 mg / kg to 10 mg / kg, approximately 6 mg / kg to 10 mg / kg, approximately 6.5 mg / kg to 10 mg / kg, approximately 7 mg / kg to 10 mg / kg, approximately 7.5 mg / kg to 10 mg / kg, approximately 8 mg / kg to 10 mg / kg, approximately 8.5 mg / kg to 10 mg / kg, approximately 9 mg / kg to 10 mg / kg, or approximately 9.5 mg / kg to 10 mg / kg. Intermediate values ​​and ranges of the stated values ​​are also intended to be part of the present invention.

[0031] For example, dsRNA is 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 It may be administered in doses of 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 approximately 10 mg / kg. Intermediate values ​​and ranges of the values ​​listed are also intended to be part of the present invention.

[0032] In another embodiment, dsRNA was approximately 0.5 to 50 mg / kg, approximately 0.75 to 50 mg / kg, approximately 1 to 50 mg / kg, approximately 1.5 to 50 mg / kb, approximately 2 to 50 mg / kg, approximately 2.5 to 50 mg / kg, approximately 3 to 50 mg / kg, approximately 3.5 to 50 mg / kg, approximately 4 to 50 mg / kg, approximately 4.5 to 50 mg / kg, approximately 5 to 50 mg / kg, approximately 7.5 to 50 mg / kg, approximately 10 to 50 mg / kg, approximately 15 to 50 mg / kg, approximately 20 to 50 mg / kg, approximately 20 to 50 mg / kg, approximately 25 to 50 mg / kg, approximately 25 to 5 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, 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 in doses of approximately 5-30 mg / kg, approximately 10-30 mg / kg, approximately 15-30 mg / kg, approximately 20-30 mg / kg, approximately 20-30 mg / kg, approximately 25-30 mg / kg, approximately 0.5-20 mg / kg, approximately 0.75-20 mg / kg, approximately 1-20 mg / kg, approximately 1.5-20 mg / kg, approximately 2-20 mg / kg, approximately 2.5-20 mg / kg, approximately 3-20 mg / kg, approximately 3.5-20 mg / kg, approximately 4-20 mg / kg, approximately 4.5-20 mg / kg, approximately 5-20 mg / kg, approximately 7.5-20 mg / kg, approximately 10-20 mg / kg, or approximately 15-20 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention. .

[0033] For example, the target values ​​are 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, 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 doses 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 approximately 50 mg / kg may be administered. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0034] In another embodiment, the present invention provides a method for inhibiting ANGPTL3 expression in a subject. This method comprises administering a therapeutically effective amount of the present invention's dsRNA or vector to a subject, thereby inhibiting ANGPTL3 expression 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 a method for inhibiting the expression of the ANGPTL3 gene in cells by contacting cells with a double-stranded RNAi agent in an amount effective to inhibit the expression of ANGPTL3 in cells. The kit comprises the RNAi agent, instructions for use, and optionally means for administering the RNAi agent to a target. [Brief explanation of the drawing]

[0036] [Figure 1] This is a schematic diagram of the experimental procedure used in the in vivo test described in Example 2. [Figure 2] Panel A of Figure 2 is a graph showing the measured levels of ANGPTL3 protein in wild-type (WT) mice after treatment with the indicated iRNA or control. Panel B of Figure 2 is a graph showing the measured levels of ANGPTL3 protein in ob / ob mice after treatment with the indicated iRNA or control. [Figure 3] Panel A of Figure 3 is a graph showing the measured levels of LDL-c in wild-type (WT) mice after treatment with the indicated iRNA or control. Panel B of Figure 3 is a graph showing the measured levels of LDL-c in ob / ob mice after treatment with the indicated iRNA or control. [Figure 4] Panel A of Figure 4 is a graph showing the measured levels of triglycerides in wild-type (WT) mice after treatment with the indicated iRNA or control. Panel B of Figure 4 is a graph showing the measured levels of triglycerides in ob / ob mice after treatment with the indicated iRNA or control. [Figure 5]Panel A of Figure 5 is a graph showing the measured levels of total cholesterol (TC) in wild-type (WT) mice after treatment with the indicated iRNA or control. Panel B of Figure 5 is a graph showing the measured levels of total cholesterol (TC) in ob / ob mice after treatment with the indicated iRNA or control. [Figure 6] Panel A of Figure 6 is a graph showing the measured levels of HDL-c in wild-type (WT) mice after treatment with the indicated iRNA or control. Panel B of Figure 6 is a graph showing the measured levels of HDL-c in ob / ob mice after treatment with the indicated iRNA or control. [Figure 7] This graph shows the measured levels of ANGPTL3 protein in human PCS transgenic mice after treatment with a single dose of the indicated iRNA or control. [Modes for carrying out the invention]

[0037] The present invention provides an iRNA composition that induces RNA-induced silencing complex (RISC) cleavage of the RNA transcript of the ANGPTL3 gene. The ANGPTL3 gene may be present in cells, for example, in subjects such as humans. The present invention also provides methods of using the iRNA composition of the present invention to inhibit the expression of the ANGPTL3 gene and / or to treat subjects suffering from lipid metabolism disorders, such as hyperlipidemia or hypertriglyceridemia, for which inhibiting or reducing the expression of the ANGPTL3 gene may be beneficial.

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

[0039] The following detailed description discloses methods for preparing and using compositions containing iRNA for inhibiting the expression of the ANGPTL3 gene, as well as compositions and methods for treating subjects suffering from diseases and disorders in which inhibition and / or reduction of the expression of this gene may be beneficial.

[0040] I. Definition To make the present invention easier to understand, several terms are first defined. Furthermore, it should be noted that whenever a variable value or range of values ​​is stated, intermediate values ​​and ranges of the stated values ​​are also intended to be part of the present invention.

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

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

[0043] The term "ANGPTL3" refers, unless otherwise specified, to angiopoietin-like protein 3 having an amino acid sequence derived from any vertebrate or mammalian source, including but not limited to humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs. 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 unprocessing precursor form of ANGPTL3, as well as the mature form obtained from post-translational cleavage of the signal peptide and the form obtained from proteolytic treatment of the fibrinogen-like domain. The sequence of the human ANGPTL3 mRNA transcript can be found, for example, at GenBank registry number GI:41327750 (NM_014495.2; SEQ ID NO: 1). The predicted sequence of rhesus monkey ANGPTL3 mRNA can be found, for example, at GenBank registry number GI:297278846 (XM_001086114.2; SEQ ID NO: 2). The sequence of mouse ANGPTL3 mRNA can be found, for example, at GenBank registry number GI:142388354 (NM_013913.3; SEQ ID NO: 3). The sequence of rat ANGPTL3 mRNA can be found, for example, at GenBank registry number GI:68163568 (NM_001025065.1; SEQ ID NO: 4).

[0044] As used herein, the term “ANGPTL3” also refers to specific polypeptides expressed in cells due to mutations in the native DNA sequence of the ANGPTL3 gene, such as single nucleotide polymorphisms (SNPs) in the ANGPTL3 gene. Many SNPs in the ANGPTL3 gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp). Non-exclusive examples of SNPs in the ANGPTL3 gene can be found in NCBI dbSNP registry numbers rs193064039;rs192778191;rs192764027;rs192528948;rs191931953;rs191293319;rs191171206;rs191145608;rs191086880;rs191012841; or rs190255403.

[0045] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the ANGPTL3 gene, including mRNA, which 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 act as a substrate for iRNA-directed cleavage at or near the corresponding portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the ANGPTL3 gene.

[0046] The target sequence can be approximately 9-36 nucleotides long, for example, approximately 15-30 nucleotides long. For example, the target sequence could be approximately 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 nucleotide lengths 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. Intermediate ranges and lengths between those listed above are also considered to be part of the present invention.

[0047] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides represented by a sequence shown using standard nucleotide nomenclature.

[0048] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it will be understood that the terms "ribonucleotide" or "nucleotide" may also refer to modified nucleotides or surrogate replacement moieties, as will be further detailed below. Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be substituted by other moieties without significantly altering the base-pairing properties of oligonucleotides containing such substitution moieties. For example, but not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine may be substituted, for example, with nucleotides containing inosine in the nucleotide sequences of dsRNAs addressed in this invention. In another example, an adenine and a cytosine molecule in an oligonucleotide may be substituted with guanine and uracil, respectively, to form a GU fluctuation base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods described in the present invention.

[0049] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferant,” as used synonymously herein, refer to agents containing RNA and mediating targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as defined herein. iRNAs induce sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate (e.g., inhibit) the expression of ANGPTL3 in cells, for example, in mammalian subjects.

[0050] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as an ANGPTL3 target mRNA sequence, to lead to the cleavage of the target RNA. Although we do not wish to be constrained by theory, a long double-stranded RNA introduced into a cell is degraded into siRNA 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). Next, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Herein, in one embodiment, the present invention relates to a single-stranded RNA (siRNA) that is generated in cells and promotes the formation of a RISC complex resulting in the silencing of a target gene, namely the ANGPTL3 gene. Accordingly, 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 the sequence in the target mRNA. The antisense RNA can stoichiometrically inhibit translation by base-pairing with the mRNA and physically interfering with the translation mechanism (see Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355). The single-stranded antisense RNA molecule is about 13 to about 30 nucleotides long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule may contain a sequence of at least about 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 herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are shown to have “sense” and “antisense” orientations toward the target RNA, i.e., the ANGPTL3 gene. In one embodiment of the present invention, double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0053] The double-stranded region may be of any length that allows for the specific degradation of the desired target RNA via the RISC pathway, and can range from approximately 9 to 36 base pairs in length, for example, in the range of approximately 15 to 30 base pairs, for example, approximately 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 lengths may be approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs, such as 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths between those described above are also considered to be part of the present 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 therefore joined by a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, the joining RNA strands are called a “hairpin loop.” A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 23 or more unpaired nucleotides.

[0055] If two substantially complementary strands of dsRNA are contained by separate RNA molecules, these molecules may or may not be covalently linked. If the two strands are covalently linked by means other than a contiguous chain of nucleotides between the 3' end of one strand and the 5' end of the other, the linking structure is called a "linker." RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus the overhangs present in the double helix. In addition to the double helix structure, RNAi may contain 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 if the 3' end of one strand of a dsRNA extends over the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of a nucleotide / nucleoside analog containing a deoxynucleotide / nucleoside. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located at the 5' end, the 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0057] In one embodiment, the antisense strand of the dsRNA has overhangs of 1 to 10 nucleotides at its 3' and / or 5' ends, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhangs are substituted with nucleoside thiophosphates.

[0058] As used herein in relation to dsRNA, the terms “blunt” or “blunt-ended” mean that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA; that is, there are no nucleotide overhangs. One or both ends of a dsRNA may be blunt. If both ends of a dsRNA are blunt, it is said to be blunt-ended. For clarity, a “blunt-ended” dsRNA is one in which both ends are blunt, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded along its entire length.

[0059] The terms “antisense strand” or “guide strand” refer to the strand of iRNA, e.g., dsRNA, that contains a region substantially complementary to the target sequence, e.g., ANGPTL3 mRNA. As used herein, the term “complementary region” refers to a region of the antisense strand that is substantially complementary to the sequence, e.g., the target sequence, e.g., ANGPTL3 nucleotide sequence, as defined herein. If the complementary region is not perfectly complementary to the target sequence, mismatches may exist in the internal or terminal regions of the molecule. Generally, most acceptable mismatches are located in terminal regions, e.g., in the 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the iRNA.

[0060] As used herein, the terms “sense strand” or “passenger strand” refer to a strand of iRNA that contains a region substantially complementary to the antisense strand, as defined herein.

[0061] As used herein, the term “complementary,” unless otherwise specified, means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under given conditions to form a double-stranded structure, where the first nucleotide sequence is described in relation to a second nucleotide sequence. 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, 50°C or 70°C for 12–16 hours, followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions, that may 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 the two sequences, depending on the final use of the hybridized nucleotides.

[0062] In iRNA, for example, in dsRNA as described herein, complementary sequences include base pairings of one or both nucleotide sequences over the full length of one or both nucleotide sequences of an oligonucleotide or polynucleotide containing a first nucleotide sequence to an oligonucleotide or polynucleotide containing a second nucleotide sequence. Such sequences may be referred to herein as “fully complementary” to each other. However, if the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be fully complementary, or, when hybridized to double helixes of up to 30 base pairs while retaining their ability to hybridize under optimal conditions for their final application, e.g., inhibition of gene expression via the RISC pathway, they may form one or more mismatched base pairs, but generally five or fewer, four or fewer, three or fewer, or two or fewer. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, for the purposes described herein, a dsRNA comprising one oligonucleotide of 21 nucleotides and the other oligonucleotide of 23 nucleotides may be referred to as “fully complementary” even if the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide.

[0063] As used herein, “complementary” sequences may include, or may be entirely formed from, non-Watson-Crick base pairs, and / or base pairs formed from non-natural and modified nucleotides, insofar as the above requirements related to their hybridizing ability are met. Such non-Watson-Crick base pairs include, non-limitingly, G:U fluctuations or Hoogstein base pairs.

[0064] In this specification, the terms “complementary,” “fully complementary,” and “substantially complementary” may be used in relation to matching bases between the sense strand and antisense strand of a dsRNA, or between the antisense strand and target sequence of an iRNA, as can be understood from the context in which they are used.

[0065] As used herein, a polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding ANGPTL3). For example, a polynucleotide is complementary to at least a portion of ANGPTL3 mRNA if its sequence is substantially complementary to a contiguous portion of the mRNA encoding ANGPTL3.

[0066] Generally, the majority of nucleotides in each chain are ribonucleotides, but as described in detail herein, each or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, “iRNA” may contain ribonucleotides having chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any such modification used in an iRNA molecule is encompassed by “iRNA” for the purposes of this specification and the claims.

[0067] As used herein, the term “inhibit” is synonymous with “reduce,” “silence,” “downcontrol,” “suppress,” and other similar terms, and includes any level of inhibition.

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

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

[0070] ANGPTL3 gene expression can be assessed based on the levels of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA levels or ANGPTL3 protein levels. ANGPTL3 expression can also be indirectly assessed 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 assessed by a decrease in the absolute or relative levels of one or more of these variables compared to control levels. Control levels may be levels of any type of control used in the art, such as pre-dose baseline levels, or levels measured from similar subjects, cells, or samples that have been untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

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

number

[0072] As used herein, the phrase “contacting an RNAi agent with cells,” such as dsRNA, includes contacting cells by any possible means. The step of contacting an RNAi agent with cells includes the step of contacting cells with iRNA in vitro or the step of contacting cells with iRNA in vivo. Contact may be direct or indirect. For example, an RNAi agent may be physically contacted with cells by performing the method individually, or the RNAi agent may be made available for subsequent contact with cells or placed in a situation that allows for subsequent contact with cells.

[0073] The process of contacting cells in vitro may be carried out, for example, by incubating the cells with an RNAi agent. The process of contacting cells in vivo may be carried out, 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, the bloodstream or subcutaneous space, so that the RNAi agent subsequently reaches the tissue where the cells being contacted are located. For example, the RNAi agent may contain and / or be bound to a ligand that directs the RNAi agent to a site of interest, for example, the liver, such as GalNAc3. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may also be contacted with an RNAi agent in vitro and then transplanted into a subject.

[0074] In one embodiment, the step of contacting cells with iRNA includes the step of “introducing” or “delivering iRNA into cells” by promoting or performing uptake or absorption into the cells. Absorption or uptake of iRNA may be carried out by unaided diffusive or active cellular processes, or by auxiliary drugs or devices. Introduction of iRNA into cells may be in vitro and / or in vivo. For example, in the case of in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be carried out by β-glucan delivery systems such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Patent Application Publication 2005 / 0281781, the entire contents of which are incorporated herein by reference. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further methods are described later in this specification and / or are known in the art.

[0075] The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP is a lipid vesicle lining a small amount of aqueous interior containing a nucleic acid, such as iRNA or a plasmid from which iRNA is transcribed. SNALPs are described, for example, in U.S. Patent Application Publication No. 20060240093, U.S. Patent Application Publication No. 20070135372, and in the brochure of the International Application, International Publication No. 2009082817, the entire contents of which are incorporated herein by reference. Examples of “SNALP” formulations are described below.

[0076] As used herein, “subject” refers to mammals, including primates (humans, non-human primates, e.g., monkeys, and chimpanzees), non-primates (cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or animals, such as birds (e.g., ducks or geese). In one embodiment, the subject is a human being treated or evaluated for a disease, disorder or condition for which a reduction in ANGPTL3 expression may be beneficial as described herein; a human being at risk of a disease, disorder or condition for which a reduction in ANGPTL3 expression may be beneficial; a human being suffering from a disease, disorder or condition for which a reduction in ANGPTL3 expression may be beneficial; and / or a human being treated for a disease, disorder or condition for which a reduction in ANGPTL3 expression may be beneficial. As used herein, the terms “treat” or “treatment” refer to beneficial or desired results, including, but not limited to, reducing triglyceride levels in the subject. The terms “to treat” or “treatment” are not limited to, but may also include, for example, the alleviation or improvement of one or more symptoms of a lipid metabolism disorder, such as a reduction in the size of xanthomas. “Treatment” may also mean an extension of survival compared to the predicted survival time without treatment.

[0077] In the context of disease markers or symptoms, “reduce” means a statistically significant reduction in such levels. The reduction may 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 considered within the normal range for individuals without such disorder. As used herein, “prevention” or “prevention” when used in relation to a disease, disorder, or condition for which a reduction in ANGPTL3 gene expression may be beneficial means that the subject is reduced to the likelihood of developing symptoms associated with such disease, disorder, or condition, such as high triglyceride levels or exanthematous xanthoma. The likelihood of developing high triglyceride levels or exanthematous xanthoma is reduced, for example, to the extent that an individual with one or more risk factors for high triglyceride levels or exanthematous xanthoma does not develop high triglyceride levels or exanthematous xanthoma, or develops milder high triglyceride levels or exanthematous xanthoma compared to a population with the same risk factors but who does not receive the treatment described herein. Effective prevention is considered to be the absence of the onset of a disease, disability, or condition, or a reduction in the incidence of symptoms associated with such disease, disability, or condition (e.g., at least about 10% on a clinically recognized scale of the disease or disability), or a delay in the onset of symptoms (e.g., by only a few days, weeks, months, or years).

[0078] As used herein, the term “serum lipids” refers to any major lipid present in the blood. Serum lipids may be present in the blood in free form or as part of protein complexes, such as lipoprotein complexes. Non-exclusive 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 associated with or caused by a lipid metabolism disorder. For example, the term includes any disorder, disease or condition that may lead to hyperlipidemia, or a condition characterized by an abnormally elevated level of any or all lipids and / or lipoproteins in the blood. The term also refers to genetic disorders such as familial hypertriglyceridemia, or acquired disorders such as those resulting from dietary habits or the intake of certain medications. Examples of lipid metabolism disorders include, but are not limited to, atherosclerosis, hyperlipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcoholic hypertriglyceridemia, β-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 chylosis, apoE deficiency or resistance, LPL deficiency or dysfunction, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, and xanthomatous (subcutaneous cholesterol deposition).

[0080] Cardiovascular diseases associated with lipid metabolism disorders are also considered “lipid metabolism disorders” as defined herein. These diseases may include coronary artery disease (also known as ischemic heart disease), inflammation, restenosis, peripheral vascular disease, and stroke associated with coronary artery disease.

[0081] Weight-related disorders are also considered “lipid metabolism disorders” as defined herein. Such disorders may include obesity, metabolic syndrome including its independent components (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 the risk of weight regrowth after weight loss.

[0082] Blood glucose disorders are further considered “lipid metabolism disorders” as defined herein. Such disorders may include diabetes mellitus, hypertension, and polycystic ovary syndrome associated with insulin resistance. Other exemplary lipid metabolism disorders may include renal transplantation, nephrotic syndrome, Cushing’s syndrome, acromegaly, systemic lupus erythematosus, abnormal globulinemia, lipodystrophy, glycogen storage disease type 1, and Addison’s disease.

[0083] As used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to treat a lipid metabolism disorder in a subject (for example, by reducing, improving, or maintaining the symptoms of a pre-existing disease or one or more of the symptoms of the disease). The “therapeutic dose” may vary depending on the RNAi agent, the method of administration, the disease and its severity and medical history, age, weight, family history, genetic structure, the type of previous or concomitant treatments, if any, and other individual characteristics of the subject being treated.

[0084] As used herein, “a prophylactically effective dose” is intended to contain an amount of iRNA sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject suffering from a lipid metabolism disorder. Improvement of the disease includes delaying the course of the disease or reducing the severity of any subsequent onset of the disease. “A prophylactically effective dose” may vary depending on the iRNA, the method of administration of the drug, the risk of developing the disease, and the medical history, age, weight, family history, genetic structure, the type of previous or concomitant treatments, if any, and other individual characteristics of the subject being treated.

[0085] The “therapeutic dose” or “prophylactically effective dose” also includes the amount of RNAi agent that produces the desired local or systemic effect, which is a reasonable benefit / risk ratio applicable to any treatment. The iRNA used in the method of the present invention may be administered in an amount sufficient to obtain a reasonable benefit / risk ratio applicable to such treatment.

[0086] The phrase "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within reasonable medical judgment and commensurate with a reasonable benefit-risk ratio, is suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0087] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in transporting or carrying 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 that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Some examples of materials that can act as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) celluloses such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, and their derivatives; (4) 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, etc. (11) Glycols; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Distilled water from which pyrogenic substances have been removed; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer; (21) Polyesters, polycarbonates, and / or polyanhydrides; (22) Expanders such as polypeptides and amino acids; (23) Serum components such as serum albumin, HDL, and LDL; and (22) Other non-toxic, suitable substances used in pharmaceutical preparations.

[0088] As used herein, the term “sample” includes similar bodily fluids, cells, or tissues isolated from a subject, as well as aggregates of bodily fluids, cells, or tissues present in the subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples derived from tissue, organ, or local area. For example, a sample may originate from a specific organ, part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may originate from the liver (e.g., the whole liver or a specific part of the liver, or a specific type of cell in the liver, such as hepatocytes). In some embodiments, “sample derived from subject” refers to blood or plasma obtained from the subject.

[0089] II. The iRNA of the present invention iRNAs that inhibit the expression of the ANGPTL3 gene are described herein. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the ANGPTL3 gene in cells, such as cells in a target, e.g., a mammal (e.g., a human suffering from a lipid metabolism disorder such as familial hyperlipidemia). The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the ANGPTL3 gene, the complementary region being 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). When in contact with cells expressing the ANGPTL3 gene, the iRNA inhibits the expression of the ANGPTL3 gene (e.g., the ANGPTL3 gene in humans, primates, non-primates, or birds) by at least approximately 10% when assayed using protein-based methods such as PCR or branched DNA (bDNA) assay, or immunofluorescence analysis using Western blotting or flow cytometry.

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

[0091] Generally, double-stranded structures are 15-30 base pairs long, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-2 The lengths are 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. Intermediate ranges and lengths between those listed above are also considered to be part of the present invention.

[0092] Similarly, regions complementary to the target sequence are 15-30 nucleotides long, 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 The nucleotide lengths are 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. Intermediate ranges and lengths between those listed above are also considered to be part of the present invention.

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

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

[0095] The dsRNAs described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. Overhangs may be located on the sense strand, antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

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

[0097] The iRNA compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of a double-stranded RNA molecule are prepared separately. Next, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase, solid-phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides can be readily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution-phase, 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 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 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 contained in separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained in one oligonucleotide.

[0099] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of approximately 20–23 base pairs, for example, 21 base pairs, have been shown to be particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, the properties of the oligonucleotide sequences shown in Tables 2, 3, 7, 8, 9 and 10 may allow the dsRNAs described herein to contain at least one strand of a minimum length of 21 nucleotides. It can be naturally predicted that shorter double-stranded DNA having one of the sequences in Tables 2, 3, 7, 8, 9, and 10, with a very small number of nucleotides subtracted from one or both ends, may be equally effective compared to the above-mentioned dsRNAs. Therefore, dsRNAs having 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 whose ability to inhibit ANGPTL3 gene expression differs from that of dsRNAs with the complete sequence by approximately 5, 10, 15, 20, 25, or 30% or less, are considered to be within the scope of the present invention.

[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. Therefore, the present invention further features iRNAs that target one of these sites. When used herein, an iRNA is considered to target a specific site within the RNA transcript if it induces cleavage of the transcript at any location within that particular site. Such an iRNA would generally consist of at least about 15 consecutive nucleotides from one of the sequences shown in Tables 2, 3, 7, 8, 9, and 10, which are ligated to a further nucleotide sequence taken from a region adjacent to a selected sequence in the ANGPTL3 gene.

[0101] Target sequences are generally about 15–30 nucleotides long, but the suitability of specific sequences within this range to guide the cleavage of any given target RNA varies. While the various software packages and guidelines described herein provide guidance for identifying the optimal target sequence for any given gene target, empirical methods can also be employed, where a “window” or “mask” of a given size (21 nucleotides as an example) is placed literally or figuratively (including in silico) on the target RNA sequence to identify sequences within a size range that can function as target sequences. The next potential target sequence can be identified by gradually shifting the sequence “window” one nucleotide upstream or downstream of the initial target sequence position until a complete set of possible sequences is identified for any given target size of choice. This process, along with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify the optimally functioning sequence, can identify the RNA sequence that best mediates the inhibition of target gene expression when targeted with an iRNA agent. Therefore, for example, the sequences identified in Tables 2, 3, 7, 8, 9, and 10 represent effective target sequences, but further optimization of inhibition efficiency can be achieved by gradually "shifting the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0102] Furthermore, for example, further optimization can be achieved 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 by testing the resulting sequences by shifting the longer or shorter size window upward or downward from that point towards the target RNA. In addition, the efficiency of inhibition can be further improved by combining this method for generating novel candidate targets in inhibition assays known in the art and / or described herein with testing the efficacy of iRNAs based on those target sequences. Moreover, such optimized sequences can be modified, 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., increased serum stability or circulating half-life, increased thermal stability, improved membrane permeable delivery, targeting to specific locations or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes).

[0103] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatched region is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is confined to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, for a 23-nucleotide iRNA agent, the strand complementary to the ANGPTL3 gene region generally contains no mismatches in the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an iRNA containing a mismatch with the target sequence is effective in inhibiting the expression of the ANGPTL3 gene. Considering the effectiveness of an iRNA with a mismatch in inhibiting the expression of the ANGPTL3 gene is particularly important when it is known that a particular complementary region in the ANGPTL3 gene has polymorphic sequence variation within the population.

[0104] III. Modified iRNA of the present invention In one embodiment, the RNA of the iRNA of the present invention, for example, dsRNA, is chemically modified to improve stability or other beneficial properties. The nucleic acids addressed in the present invention may be synthesized and / or modified by methods well established in the art, such as those described herein by reference as “Current protocols in nucleic acids chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugate, inverted linkage) or 3'-terminal modifications (conjugate, DNA nucleotide, inverted linkage, etc.); base modifications, e.g., substitution with stable bases, unstable bases, or bases that base-pair with a wide range of partners, base removal (non-basic nucleotides), or conjugate bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or skeletal modifications, including modification or substitution of phosphodiester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing a modified skeleton or RNAs that do not contain natural internucleoside bonds. RNAs having a modified skeleton include, in particular, those that do not have a phosphorus atom in the skeleton. For the purposes of this specification and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in the internucleoside skeleton can also be considered as oligonucleosides. In some embodiments, the modified iRNA has a phosphorus atom in its internucleoside skeleton.

[0105] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoamides and aminoalkylphosphoamides, thionophosphoamides, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates having the usual 3'-5' linkage, their 2'-5' linkage analogs, and those with inverted polarity where pairs of adjacent nucleoside units are linked to 3'-5'~5'-3' or 2'-5'~5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0106] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above include, but are not limited to, U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; and No. 5,32 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, Examples include U.S. Patent No. 639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of these are incorporated herein by reference.

[0107] Modified RNA backbones that do not contain phosphorus atoms internally have backbones formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include those having morpholino bonds (partially formed 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 having mixed N, O, S, and CH2 constituent parts.

[0108] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patent No. 5,034,506; No. 5,166,315; No. 5,185,444; No. 5,214,134; No. 5,216,141; No. 5,235,033; No. 5,64,562; No. 5,264,564; No. 5,405,938; No. 5,434,257; No. 5,466,677; No. 5,470,967; and Examples include Specification No. 5,489,677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704; No. 5,623,070; No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439, the entire contents of each of these are incorporated herein by reference.

[0109] In other embodiments, suitable RNA mimetic compounds are considered for use in iRNA, where both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic compound, that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and directly or indirectly bonded to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; No. 5,714,331; and No. 5,719,262, the entire contents of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0110] Some embodiments of the present invention include RNA having a phosphorothioate skeleton and oligonucleosides having a heteroatom skeleton, in particular including the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene (methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [wherein the natural phosphodiester skeleton is represented as --O--P--O--CH2--], and the amide skeleton of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, the RNA described herein has the morpholino skeleton structure of the aforementioned U.S. Patent No. 5,034,506.

[0111] The modified RNA may also contain one or more substituted sugar moieties. The iRNAs taken up herein, for example, dsRNAs, can contain at the 2'-position one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA has at the 2'-position C1-C 10The modifications include lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalator groups, groups that improve the pharmacokinetic properties of iRNA, or groups that improve the pharmacodynamic properties of iRNA, and one of other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications are 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the following examples herein.

[0112] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions in the iRNA's RNA, particularly on the 3' terminal nucleotide or at the 3' and 5' positions of the sugar in the 2'-5' linked dsRNA. The iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; Nos. 5,118,800; Nos. 5,319,080; Nos. 5,359,044; Nos. 5,393,878; Nos. 5,446,137; Nos. 5,466,786; Nos. 5,514,785; Nos. 5,519,134; and Nos. 5,567,81 Examples include Specification No. 1; Specification No. 5,576,427; Specification No. 5,591,722; Specification No. 5,597,909; Specification No. 5,610,300; Specification No. 5,627,053; Specification No. 5,639,873; Specification No. 5,646,265; Specification No. 5,658,873; Specification No. 5,670,633; and Specification No. 5,700,920, some of which are owned by the same person as the present application. The entire contents of each of the above are incorporated herein by reference.

[0113] iRNA may also include nucleotide base modifications or substitutions (often simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleotide bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleotide bases 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 (pseudracil), 4 -Includes 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 (daazaguanin), and other synthetic and natural nucleic acid bases such as 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in 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 nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. The 5-methylcytosine substituent has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276–278), and is an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0114] Representative U.S. patents teaching the above-mentioned modified nucleic acid bases and several other modified nucleic acid base preparations include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; and 5,594,1 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 Examples include the following specifications: Nos. 6,380,368; Nos. 6,528,640; Nos. 6,639,062; Nos. 6,617,438; Nos. 7,045,610; Nos. 7,427,672; and Nos. 7,495,088, the entire contents of each of these specifications incorporated herein by reference.

[0115] The RNA of iRNA can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety, which contains additional crosslinks connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in its 3'-endo structural configuration. Adding 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 U.S. patents teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent 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 which are incorporated herein by reference.

[0117] Potentially stable modifications to the ends of RNA molecules may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3”-phosphate, and inverted base dT (idT). Disclosure of these modifications can be found in PCT publication number International Publication No. 2011 / 005861.

[0118] IV. IRNA bound to ligand Another modification of the iRNA of the present invention involves chemically attaching one or more ligands, portions, or conjugates to the RNA to improve the activity, cell distribution, or cell uptake of the iRNA. These parts are not limited to, but include the cholesterol portion (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, for example, beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NYAcad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), and thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res.,20:533-538), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,(1995)Tetrahedron Lett.,36:3651-3654; Shea et al.,(1990)Nucl.Acids Res., 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmityl moiety (Mishra et al., (1995) Biochim. Biophys.Examples include lipid moieties such as Acta, 1264:229-237, or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0119] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides, for example, improved affinity to selected targets (e.g., molecules, cells, or cell types), compartments (e.g., compartments of cells or organs), body tissues, organs, or regions compared to species without such ligands. Preferred ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0120] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-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 acid anhydride copolymer, poly(L-lactide-coglycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helix peptides.

[0121] The ligand may also include a target group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein, or an antibody that binds to a specific cell type, such as kidney cells. The target group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0122] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid (cholenic acid Examples include acids, dimethoxytrityl, or phenoxazine, and peptide complexes (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole complex, Eu3+ tetraaza macrocyclic complex), dinitrophenyl, HRP, or AP.

[0123] Ligands can be proteins, such as glycoproteins, or peptides, such as coligands, or antibodies, such as antibodies that bind to specific cell types, including hepatocytes. Ligands may also include hormones and hormone receptors. Ligands may also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species such as polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands may be, for example, lipopolysaccharides, activators of 38MAP kinase, or activators of NF-κB.

[0124] A ligand can be a substance, such as a drug, that can improve the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton, such as by disrupting the cellular microtubules, microfilaments, and / or intermediate filaments. The drug may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

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

[0126] The ligand-conjugated oligonucleotides of the present invention can be synthesized using oligonucleotides having reactive pendant functional groups, such as those derived from the binding of a binding molecule to the oligonucleotide (as described below). These reactive oligonucleotides may react directly with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands to which the binding moiety is bound.

[0127] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means known in the art for such synthesis may be used in addition to or instead of these. Similar techniques for preparing other oligonucleotides, such as phosphorothioates and alkylated derivatives, are also known.

[0128] In the ligand-conjugate oligonucleotides and ligand molecules having sequence-specific binding nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled in a suitable DNA synthesis apparatus using standard nucleotide or nucleoside precursors, nucleotide or nucleoside conjugate precursors already having a binding site, ligand-nucleotide or nucleoside conjugate precursors already having a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0129] When using a nucleotide conjugate precursor that already has a binding site, the synthesis of the sequence-specific binding nucleoside is usually completed first, after which the ligand molecule reacts with the binding site to form a ligand-conjugate oligonucleotide. In one embodiment, the oligonucleotide or binding nucleoside of the present invention is synthesized by an automated synthesizer using commercially available standard and non-standard phosphoramidites commonly used in oligonucleotide synthesis, as well as phosphoramidites derived from ligand-nucleoside conjugates.

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

[0131] The binding of conjugates to target tissues can be inhibited, for example, controlled, using lipid-based ligands. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. Conjugates can be targeted to the kidneys using lipids or lipid-based ligands that bind more weakly to HSAs.

[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 is distributed to non-renal tissue. 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 to or does not bind at all to the HSA so that the conjugate is preferably distributed to the kidney. Other portions targeting renal cells may also be used instead of or in addition to the lipid-based ligand.

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

[0135] B. Cell permeability agents In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, this agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If this agent is a peptide, it may be modified by including the use of peptidyl mimes, inverted isomers, non-peptide or pseudopeptide bonds, and D-amino acids. The helical agent is preferably an α-helix agent, which preferably has lipophilic and oleophobic phases.

[0136] The ligand may be a peptide or a peptide mimetic. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules that can fold into a distinct three-dimensional structure similar to that of natural peptides. Binding of peptides and peptide mimes to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by improving cell recognition and absorption. The peptide or peptide mimetic portion may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0137] The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., mainly consisting of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative example, the peptide moiety may contain a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be target moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been shown to function as delivery peptides. Peptides or peptide mimetic compounds 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.). (al., Nature, 354:82-84, 1991). Examples of peptides or peptide mimetic compounds linked to dsRNA agents via monomer units incorporated for the purpose of cell targeting include arginine-glycine-aspartate (RGD) peptides or RGD mimetic compounds. The peptide portion may range in length from approximately 5 to approximately 40 amino acids. The peptide portion may have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below may be used.

[0138] The RGD peptides used in the compositions and methods of the present invention may be linear or cyclic, and may be modified, for example, glycosylated or methylated, to facilitate targeting of specific tissues. RGD-containing peptides and peptide mimetic compounds may include D-amino acids and synthetic RGD mimetic compounds. In addition to RGD, other parts that target integrin ligands can be used. Preferred conjugates of these ligands target PECAM-1 or VEGF.

[0139] "Cell-permeable peptides" are capable of permeating cells, such as microbial cells like bacteria or fungal cells, or mammalian cells like human cells. Peptides that permeate microbial cells may be, for example, α-helix linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenecin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphimotic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0140] C. Carbohydrate Conjugate 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 in vivo delivery of nucleic acids as described herein, and the compositions are suitable for in vivo therapeutic use. As used herein, “carbohydrate” means a compound that is a carbohydrate itself, consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each of which is bonded to an oxygen, nitrogen, or sulfur atom; or a compound that, as part thereof, has a carbohydrate moiety consisting 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 bonded to an oxygen, nitrogen, or sulfur atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gum. Certain monosaccharides include sugars with 5 or more C5 units (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0141] In one embodiment, the carbohydrate conjugate used in the composition and method of the present invention is a monosaccharide. In one embodiment, the monosaccharide is [ka] These include N-acetylgalactosamine.

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

[0143] Other representative carbohydrate conjugates for use in the embodiments described herein include, but are not limited to, [ka] (Formula XXIII) is an example, where if 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, such as PK modifiers and / or cell-permeable peptides, as described above, without limitation.

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

[0146] The term "linker" or "bonding group" refers to an organic part that connects two parts of a compound, for example, by covalently bonding two parts of a compound. Linkers are typically directly bonded, or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or non-limited substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryls, heteroaryls, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls, alkenylheteroarylalkyls, Alkenyl heteroaryl alkenyl, alkenyl heteroaryl alkynyl, alkynyl heteroaryl alkyl, alkynyl heteroaryl alkenyl, alkynyl heteroaryl alkynyl, alkyl heterocyclyl alkyl, alkyl heterocyclyl alkenyl, alkyl hetero(herero)cyclyl alkynyl, alkenyl heterocyclyl alkyl, alkenyl heterocyclyl alkenyl, alkenyl heterocyclyl alkynyl, alkynyl heterocyclyl alkyl, alkynyl heterocyclyl alkenyl The compounds comprise a chain of atoms such as nyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylhetero(herero)aryl (where one or more methylene atoms may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic); where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker consists of approximately 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 17, or 8 to 16 atoms.

[0147] The cleavable binding group is sufficiently stable outside the cell, but after entering the target cell, it is cleaved, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable binding group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, or at least about 100 times faster, in the target blood or under a first reference condition (which may be selected to mimic or represent intracellular conditions) or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0148] Bindable binding groups are sensitive to the presence of cleavage agents, such as pH, redox potential, or degradable molecules. Generally, cleavage agents are more widely present in cells than in serum or blood, or are found at higher levels or activity. Examples of such degrading agents include: oxidizing or reductases present in cells, or reducing agents such as mercaptans that can degrade redox-cleavable binding groups by reduction, selected for specific substrates or lacking substrate specificity; esterases; agents that can create endosomes or acidic environments, for example, by providing a pH of 5 or less; enzymes, peptidases (which may be substrate-specific), and phosphatases that can hydrolyze or degrade acid-cleavable binding groups by acting as general acids.

[0149] Cleavable binding groups, such as disulfide bonds, can be pH sensitive. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers, by having cleavable binding groups that are cleaved at a favorable pH, will release cationic lipids from ligands within the cell or into desired compartments of the cell.

[0150] The linker may contain cleavable binding groups that can be cleaved by specific enzymes. The type of cleavable binding group incorporated into the linker may depend on the target cell. For example, a ligand targeting the liver may be bound to cationic lipids via a linker containing an ester group. Because hepatocytes are rich in esterases, this linker will be cleaved more efficiently within hepatocytes than within esterase-deficient cell types. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

[0151] Linkers containing peptide bonds can be used to target peptidase-rich cell types, such as hepatocytes and synovial cells.

[0152] Generally, the suitability of a candidate cleavable binding group can be evaluated by testing the ability of a degradation agent (or degradation condition) to cleave the candidate binding group. It is also desirable to test the ability of the candidate cleavable binding group to resist cleavage in the blood or in contact with other non-target tissues. Therefore, the relative sensitivity to cleavage between the first and second conditions can be determined, with the first condition selected to demonstrate cleavage within target cells and the second condition selected to demonstrate cleavage in other tissues or biological fluids, such as blood or serum. This evaluation can be performed in a cell-free system, intracellularly, in cell cultures, in organs or tissue cultures, or in whole animals. It may be useful to perform the initial evaluation in cell-free or culture conditions and confirm it with further evaluation in animals. 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 intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0153] i. Cleavable redox bond groups In one embodiment, the cleavable binding group is a redox-cleavable binding group that is cleaved after reduction or oxidation. An example of a reductively cleavable binding group is a disulfide binding group (-SS-). To determine whether a candidate cleavable binding group is a suitable "reductively cleavable binding group" or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent, the methods described herein may be consulted. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which mimics the rate of cleavage that would be observed in cells, e.g., target cells. A candidate can also be evaluated under conditions selected to mimic blood or serum conditions, in which case the candidate compound is cleaved by up to about 10% in blood. In another embodiment, a useful candidate compound is degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using a standard enzyme kinetics assay under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0154] ii. Phosphate-based cleavable bonding groups In another embodiment, the cleavable linker includes a phosphate-based cleavable binding group. The phosphate-based cleavable binding group is cleaved by agents that decompose or hydrolyze the phosphate group. An example of an agent that cleaves the phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based binding groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

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

[0156] iv. Ester-based cleavable bond groups In another embodiment, the cleavable linker includes an ester-based cleavable binding group. The ester-based cleavable binding group is cleaved by enzymes such as esterases and amylases in cells. Examples of ester-based cleavable binding groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester-cleavable binding group has 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 binding groups 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, which is formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond, which is formed between amino acids to give peptides and proteins. The peptide-based cleavable group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, and does not include all amide functional groups. The peptide-based cleavable linking group 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 with a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrates conjugated with the linker of the composition and method of the present invention include: [ka] [ka] [ka] This includes the following: When one of X or Y is an oligonucleotide, the other is hydrogen.

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

[0160] In one embodiment, the dsRNA of the present 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] In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent values ​​from 0 to 20 for each existence, and the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C Each of these is independently absent with respect to each presence, 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 These are, independently with respect to each presence, absent, alkylene, or substituted alkylene, where one or more methylene are O, S, S(O), SO2, N(R) N ), may be interrupted or terminated by one or more of the following: C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5CEach is independently absent with respect to its respective existence: NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocycline; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C R represents a ligand; that is, each is independently a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide in relation to each other; a is either H or an amino acid side chain. Trivalent conjugated GalNAc derivatives, such as those of formula (XXXV), are particularly useful when used with RNAi agents to inhibit the expression of target genes: [ka] In the formula, L 5A , L 5B and L 5C This represents monosaccharides such as GalNAc derivatives.

[0161] Suitable examples of divalent and trivalent branched linkage groups for conjugation to GalNAc derivatives include, but are not limited to, the structures referenced above as formulas II_VII, XI, X, and XIII.

[0162] Representative patents teaching the preparation of RNA conjugates include, without limitation, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; and 5,118,80 U.S. Patent No. 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 U.S. Patent No. 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, Specification No. 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. 5,416,203,5,451,463; U.S. Patent No. 5,510,475; U.S. Patent No. 5,512,667; U.S. Patent No. 5,514,785; U.S. Patent No. 5,565,552; U.S. Patent No. 5,567,810; U.S. Patent No. 5,574,142;U.S. Patent 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 are examples of U.S. Patents, the entire contents of each of these are incorporated herein by reference.

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

[0164] In the context of this invention, a “chimeric” iRNA compound or “chimeric” is an iRNA compound, preferably a dsRNA containing two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region, where the RNA is modified to give the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to a target nucleic acid. Further regions of the iRNA can act as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded DNA. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. Consequently, when chimeric dsRNAs are used, equivalent results are often obtained with shorter iRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of RNA targets can typically be detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0165] In some cases, the RNA of iRNA can be modified with non-ligand groups. Several non-ligand molecules are bound to iRNA to enhance its activity, cell distribution, or cell uptake, and procedures for such binding are available in the scientific literature.Such non-ligand portions 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, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992,20:533), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al.,EMBO J.,1991,10:111; Kabanov et al.,FEBS Lett.,1990,259:327; Svinarchuk et al.,Biochimie,1993,75:49), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36:3651; Shea et al.,Nucl.Acids Res.,1990,18:3777), polyamine or polyethylene glycol chains (Manoharan et al.,Nucleosides & It contained lipid moieties such as Nucleotides (1995, 14:969), adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical binding protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group is then reacted with the molecule using a suitable coupling agent or activator. The binding reaction can be performed using RNA still bound to a solid support or after cleavage of RNA in the solution phase. Purification of the RNA conjugate by HPLC usually yields a pure conjugate.

[0166] IV. Delivery of iRNA according to the present invention The iRNA of the present invention can be delivered to cells in a subject, such as a human subject (e.g., a subject requiring iRNA, such as a subject suffering from a lipid metabolism disorder), in several different ways. For example, delivery may be carried out by bringing the cells into contact with the iRNA of the present invention either in vitro or in vivo. In vivo delivery may also be carried out directly by administering a composition containing the iRNA, such as dsRNA, to the subject. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode and lead to the expression of the iRNA. These alternatives are further described below.

[0167] In general, any method for delivering nucleic acid molecules (in vitro or in vivo) may be adapted for use with the iRNA of the present invention (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, which are incorporated herein by reference in their entirety). In the case of in vivo delivery, factors to be considered for delivering the iRNA molecule include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, for example, by direct injection or transplantation into tissue, or by local administration of the formulation. Local administration to the treatment site can maximize the local concentration of the agent, limit exposure of the agent to systemic tissues that may be adversely affected or degraded by the agent, and reduce the total dose of the administered iRNA molecule. Several studies have demonstrated successful knockdown of gene products when iRNA is administered locally. For example, intravitreal injection of VEGF dsRNA into cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection into mice (Reich, SJ. et al., (2003) Mol.Vis.9:210-216) both demonstrated prevention of neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice reduced tumor volume (Pille, J. et al., (2005) Mol.Ther.11:267-274) and extended the survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol.Ther.14:343-350; Li, S. et al., (2007) Mol.Ther.15:515-523).RNA interference can be delivered locally to the central nervous system by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, 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 delivered locally to the lungs by intranasal administration (Howard, KA. et al. Success has also been demonstrated by (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). For systemic administration of iRNA for the treatment of disease, the RNA can be modified or delivered using a drug delivery system; both methods work to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or drug carriers can also enable targeting of the iRNA composition to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical binding to lipophilic groups such as cholesterol, which improves cellular uptake and prevents degradation. For example, when iRNA conjugated to the lipophilic cholesterol portion of ApoB was systemically administered to mice, knockdown of apoB mRNA was achieved in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of iRNA to aptamers has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNA may be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of negatively charged iRNA molecules and enhance interactions with negatively charged cell membranes, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may bind to iRNA or be induced to form vesicles or micelles that enclose the iRNA (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of iRNA when administered systemically. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al. (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), see above; Verma, UN. et al., (2003), see above), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111-114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of Examples include (print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In one embodiment, the iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrin can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in whole.

[0168] A. The iRNA of the present invention encoded by a vector 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. 00 / 22113, Conrad, International PCT Publication No. 00 / 22114, and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (approximately several hours to several weeks) or persistent (several weeks to several months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which may be embedded or non-embedded vectors. The introduced gene 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] Individual strands of iRNA can be transcribed from a promoter in an expression vector. If two separate strands are expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced into target cells (for example, by transfection or infection). Alternatively, each individual strand of dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, dsRNA is expressed as a reverse repeat polynucleotide joined by a linker polynucleotide sequence to have a stem-loop structure.

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

[0171] iRNA expression plasmids 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 one week or more are also envisioned in this invention. The success of vector introduction into host cells can be monitored using various known methods. For example, transient transfection can be indicated using a reporter such as a fluorescent marker such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured by using markers that confer resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance, to 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 and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors or avian pox, e.g., poxvirus vectors such as canarypox or fowlpox; and (j) helper-dependent or attenuated adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may optionally include a viral sequence for transfection. Alternatively, the construct may be incorporated into an episomal replication-capable vector, e.g., EPV and EBV vectors. Constructs for the recombinant expression of iRNA generally require regulatory elements, such as promoters and enhancers, to ensure iRNA expression within the target cell. Other embodiments of vectors and constructs are considered and will be discussed further later.

[0173] A vector useful for iRNA delivery will contain sufficient regulatory elements (promoters, enhancers, etc.) to express the iRNA in the desired target cells or tissues. These regulatory elements may be selected to provide either constitutive or regulatory / inducible expression.

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

[0175] Viral vectors containing nucleic acid sequences encoding iRNA 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 host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors that facilitate the 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 demonstrating the use of retroviral vectors in gene therapy include 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 that may be considered for use include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; No. 5,665,557; and No. 5,981,276, which are incorporated herein by reference.

[0176] Adenoviruses are also considered for use in iRNA delivery according to the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelium. Adenoviruses naturally infect respiratory epithelium and cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, (1993) Current Opinion in Genetics and Development 3:499-503 provides an overview of adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to deliver genes to rhesus monkey respiratory epithelium. Other examples of the use of adenoviruses 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 International Pamphlet No. 94 / 12649; and Wang et al., (1995) Gene Therapy 2:775-783. Suitable AV vectors for expressing the iRNAs addressed in this 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 present invention (Walsh et al., (1993) Proc.Soc.Exp.Biol.Med.204:289-300; U.S. Patent No. 5,436,146). In one embodiment, the iRNA may be expressed as two distinct, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNAs addressed in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors 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 Publication No. 94 / 13788; and International Patent Application Publication No. 93 / 24641, which are fully disclosed herein by reference.

[0178] Other viral vectors suitable for delivering the iRNA of the present invention include vaccinia viruses, such as attenuated vaccinia like Modified Virus Ankara (MVA) or NYVAC, and poxviruses such as avian poxes like fowlpox or canarypox.

[0179] The directivity of a viral vector 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, Mocola, etc. AAV vectors can be constructed to target different cells by manipulating the vector to express different capsid protein serotypes. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the full disclosure of which is incorporated herein by reference.

[0180] A vector-based pharmaceutical formulation may contain the vector in an acceptable diluent, or it may contain a sustained-release matrix into which the gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical formulation may contain one or more cells that produce the gene delivery system.

[0181] V. Pharmaceutical Composition of the Present Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier is provided herein. The iRNA-containing pharmaceutical composition is useful for treating diseases or disorders related to 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 delivery method. One example is a composition formulated for systemic administration via parenteral administration, for example, intravenous (IV) delivery or subcutaneous delivery. Another example is a composition formulated for direct delivery to the liver, for example, by infusion into the liver via a sustained-release pump.

[0183] The pharmaceutical composition of the present invention can be administered in a dose sufficient to inhibit the expression of the ANGPTL3 gene. Generally, preferred doses of the iRNA of the present invention range from about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient per day, and generally range from about 1 to 50 mg per kilogram of body weight per day. For example, dsRNA can be administered in single doses of 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.

[0184] For example, dsRNA is approximately 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 It may be administered in doses of 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 approximately 10 mg / kg. Intermediate values ​​and ranges of the values ​​listed are also intended to be part of the present invention.

[0185] In another embodiment, dsRNA is present in concentrations of approximately 0.1 to 50 mg / kg, 0.25 to 50 mg / kg, 0.5 to 50 mg / kg, 0.75 to 50 mg / kg, 1 to 50 mg / kg, 1.5 to 50 mg / kb, 2 to 50 mg / kg, 2.5 to 50 mg / kg, 3 to 50 mg / kg, 3.5 to 50 mg / kg, 4 to 50 mg / kg, 4.5 to 50 mg / kg, 5 to 50 mg / kg, 7.5 to 50 mg / kg, 10 to 50 mg / kg, 15 to 50 mg / kg, and 20 to 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, approximately 2.5 to approximately 40 mg / kg, approximately 3 to approximately 40 mg / kg, approximately 3.5 to approximately 40 mg / kg, approximately 4 to approximately 40 mg / kg, approximately 4.5 to approximately 40 mg / kg, approximately 5 to approximately 40 mg / kg, approximately 7.5 to approximately 40 mg / kg, approximately 10 to approximately 40 mg / kg, approximately 15 to approximately 40 mg / kg, approximately 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 It is administered in doses of g / kg, approximately 0.25–20 mg / kg, approximately 0.5–20 mg / kg, approximately 0.75–20 mg / kg, approximately 1–20 mg / kg, approximately 1.5–20 mg / kg, approximately 2–20 mg / kg, approximately 2.5–20 mg / kg, approximately 3–20 mg / kg, approximately 3.5–20 mg / kg, approximately 4–20 mg / kg, approximately 4.5–20 mg / kg, approximately 5–20 mg / kg, approximately 7.5–20 mg / kg, approximately 10–20 mg / kg, or approximately 15–20 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of this invention.

[0186] For example, dsRNA is 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. It may be administered in doses of 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 approximately 10 mg / kg. Intermediate values ​​and ranges of the values ​​listed are also intended to be part of the present invention.

[0187] In another embodiment, dsRNA was approximately 0.5 to 50 mg / kg, approximately 0.75 to 50 mg / kg, approximately 1 to 50 mg / kg, approximately 1.5 to 50 mg / kb, approximately 2 to 50 mg / kg, approximately 2.5 to 50 mg / kg, approximately 3 to 50 mg / kg, approximately 3.5 to 50 mg / kg, approximately 4 to 50 mg / kg, approximately 4.5 to 50 mg / kg, approximately 5 to 50 mg / kg, approximately 7.5 to 50 mg / kg, approximately 10 to 50 mg / kg, approximately 15 to 50 mg / kg, approximately 20 to 50 mg / kg, approximately 20 to 50 mg / kg, approximately 25 to 50 mg / kg, approximately 25 to 5 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, 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 in doses of approximately 5-30 mg / kg, approximately 10-30 mg / kg, approximately 15-30 mg / kg, approximately 20-30 mg / kg, approximately 20-30 mg / kg, approximately 25-30 mg / kg, approximately 0.5-20 mg / kg, approximately 0.75-20 mg / kg, approximately 1-20 mg / kg, approximately 1.5-20 mg / kg, approximately 2-20 mg / kg, approximately 2.5-20 mg / kg, approximately 3-20 mg / kg, approximately 3.5-20 mg / kg, approximately 4-20 mg / kg, approximately 4.5-20 mg / kg, approximately 5-20 mg / kg, approximately 7.5-20 mg / kg, approximately 10-20 mg / kg, or approximately 15-20 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention. .

[0188] For example, the target values ​​are 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, 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 doses 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 approximately 50 mg / kg may be administered. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0189] The pharmaceutical composition may be administered once daily, or the iRNA may be administered in two or three or more subdoses at appropriate intervals throughout the day, or further, by delivery via continuous infusion or sustained-release formulation. In this case, the amount of iRNA contained in each subdose must be correspondingly smaller in order to achieve the total daily dose. The dose unit may also be formulated for delivery over several days, for example, using conventional sustained-release formulations that provide sustained release of iRNA over a period of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites, and can therefore be used with the drug of the present invention. In this embodiment, the dose unit comprises a corresponding multiple of the daily dose.

[0190] Because the effect of a single dose on ANGPTL3 levels can be long-lasting, subsequent doses are administered at intervals of 3, 4, or 5 days or less, or at intervals of 1, 2, 3, or 4 weeks or less.

[0191] Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or illness, previous treatments, the overall health and / or age of the subject, and other pre-existing diseases, may influence the dose and time required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective amount of the composition may consist of a single treatment or a series of treatments. The effective dose and in vivo half-life for each iRNA encompassed by the present invention can be estimated using conventional methodologies or based on in vivo studies using appropriate animal models as described elsewhere in this specification.

[0192] Advances in mouse genetics have led to the creation of numerous mouse models for studying various human diseases, such as lipid metabolism disorders, where reduced ANGPTL3 expression may be beneficial. Such models can be used for in vivo testing of iRNAs, as well as for determining effective therapeutic doses. Suitable mouse models are known in the art and include, for example, obese (ob / ob) mice containing mutations in the obesity (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); mice containing homozygous knockout of the LDL receptor (LDLR- / - mice; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); a mouse model of diet-induced atherosclerosis (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 composition of the present invention may be administered in several ways, depending on whether local or systemic treatment is required and the site to be treated. Administration may be local (e.g., by a transdermal patch), pulmonary administration by inhalation or blowing of powder or aerosol, for example, using a sprayer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral administration. Parenteral administration may be intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; for example, subcutaneous administration using an implantable device; or, for example, intracranial administration by intraparenchymal, intrathecal or intraventricular administration.

[0194] iRNAs can be delivered to target specific tissues, such as the liver (e.g., hepatocytes in the liver).

[0195] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, droplets, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., are required or may be desired. Covered condoms and gloves may also be useful. Suitable topical formulations include those in which the iRNA characterizing the present invention is a mixture with a topically delivered agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleyltetramethylaminopropyl DOTAP and dioleylphosphatidylethanolamine DOTMA). The iRNAs characterizing the present invention can be encapsulated in liposomes or can form complexes with liposomes, particularly cationic liposomes. Alternatively, the iRNAs may be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaplate, tricaplate, monoolein, dilaurin, glyceryl 1-monocaplate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Examples include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0196] A. iRNA preparations containing membrane molecular assemblies iRNAs for use in the compositions and methods of the present invention can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include monolayer and multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior and usually does not contain the iRNA composition, but may in some cases. Liposomes are useful for the transport and delivery of active ingredients to the site of action. Because the liposome membrane is structurally similar to that of biological membranes, when a liposome adheres to a tissue, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the fusion of the liposome and the cell progresses, the aqueous contents inside, including the iRNA, are delivered to the cell, where the iRNA can specifically bind to target RNA and mediate RNAi. In some cases, liposomes can also be specifically targeted, for example, to direct iRNAs towards a particular cell type.

[0197] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholates, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Next, the RNAi agent preparation is added to the micelles containing the lipid component. 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 support compound to aid condensation may be added during the condensation reaction, for example, by controlled addition. For example, the support compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH may also be adjusted to aid condensation.

[0199] Methods for generating stable polynucleotide delivery vehicles incorporating polynucleotide / cationic lipid complexes as components of the delivery vehicle are further described, for example, in International Publication No. 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 This may also include one or more embodiments of the exemplary methods described in al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid assemblies of a suitable size for use as a delivery vehicle include sonication, freeze-thaw, and extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161). Microfluidization may be used when consistently small (50–200 nm) and relatively uniform assemblies are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are readily adaptable for packaging RNAi preparations 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 transported 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] pH-sensitive and negatively charged liposomes capture nucleic acids rather than complex them. Since both nucleic acids and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are captured within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to a cell monolayer in a culture medium. Expression of the exogenous 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 derived phosphatidylcholine. For example, neutral liposome compositions may be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic membrane-fused liposomes are primarily formed from dioleylphosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soy PC and egg PC. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0203] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; U.S. Patent Nos. 5,171,678; International Publication No. 94 / 00569; International Publication No. 93 / 24640; International Publication No. 91 / 16024; Felgner, (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] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been tested, and their usefulness in drug delivery to the skin has been determined. Cyclosporine-A was delivered to the dermis of mouse skin using nonionic liposome formulations containing Novasome® I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome® II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results showed that such nonionic liposome systems are effective in promoting the deposition of cyclosporine-A in different layers of the skin (Hu et al (1994). STPPharma.Sci., 4, 6, 466).

[0205] Liposomes also include “stereostabilized” liposomes, and as used herein, this term refers to liposomes containing one or more specific lipids, which, when incorporated into the liposome, result in an enhanced cyclic lifespan compared to liposomes lacking such specific lipids. An example of a stereostabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome contains (A) monosialoganglioside G M1These include (B) those containing one or more glycolipids, or those derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. Although not bound by any particular theory, in the art, with regard to sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivativeized lipids, the enhanced circulating half-life of these sterically stabilized liposomes is thought to be due to reduced intracellular uptake by the reticuloendothelial system (RES) (Allen et al., (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. NYAcad. Sci., 1987, 507, 64) developed monosialoganglioside G to improve the blood half-life of liposomes. M1 The capabilities of galactocerebroside sulfate and phosphatidylinositol have been reported. These findings are detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Both U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924, both granted to Allen et al., describe (1) sphingomyelin and (2) ganglioside G M1 Alternatively, liposomes containing galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0207] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot fuse with the cell membrane as efficiently, but they can be taken up by macrophages in vivo and used to deliver RNAi agents to macrophages.

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

[0209] Using N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a positively charged synthetic cationic lipid, small liposomes can be formed that spontaneously interact with nucleic acids, fuse with negatively charged lipids in the cell membrane of tissue culture cells, and form lipid-nucleic acid complexes capable of delivering RNAi agents (see, for example, Felgner, Plet al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Patent No. 4,897,355 for a description of DOTMA and its use with DNA).

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

[0211] Other reported cationic lipid compounds include those conjugated to one of two types of lipids, such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam®, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES"), and those conjugated to various moieties including carboxyspermine (see, for example, U.S. Patent No. 5,171,678).

[0212] Other cationic lipid conjugates include lipid derivatization with cholesterol formulated into liposomes in combination with DOPE ("DC-Chol") (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, produced 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 have been shown to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for oligonucleotide delivery are described in International Publication No. 98 / 39359 and International Publication No. 96 / 37194.

[0213] Liposome formulations are particularly well-suited for topical administration, and they offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver RNAi agents to the skin. In some implementations, liposomes are used to deliver RNAi agents to epidermal cells and to facilitate their penetration into dermal tissue, such as the skin. For example, liposomes can be applied topically. Local delivery of drugs formulated as liposomes to the skin has been reported (e.g., Weiner et al., (1992) Journal of Drug Targeting, vol.2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. Jand 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, R. Rand Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Rand See Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855.

[0214] Furthermore, nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, were investigated to determine their usefulness in drug delivery to the skin. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing RNAi agents are useful for treating skin diseases.

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

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

[0217] Transfersomes are another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can also be described as lipid droplets, which, due to their high deformability, can easily permeate smaller pores than droplets. Transfersomes are adaptable to the environment in which they are used, for example, self-optimal (adapting to the shape of pores in the skin), self-repairing, often reaching their targets without fragmentation, and frequently self-loading. To construct transfersomes, it is possible to add surface edge activators, usually surfactants, to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0218] Surfactants have found broad applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the numerous different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

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

[0220] When a surfactant molecule retains a negative charge when dissolved or dispersed in water, it is classified as anionic. Anionic surfactants include carboxylates such as soap, acyl lactylates, acylamides of amino acids, sulfate esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

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

[0222] A surfactant is classified as amphoteric if its molecule has the ability to possess either a positive or negative charge. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

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

[0224] iRNA for use in the methods of the present invention may also be provided as micelle formulations. A “micelle” is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic parts of the molecules face inward and the hydrophilic parts remain in contact with the surrounding aqueous phase. The opposite arrangement exists when the environment is hydrophobic.

[0225] Mixed micelle formulations suitable for transdermal delivery include aqueous solutions of siRNA compositions and alkali metals C8-C8. 22 It can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocolanyglycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogues, polydocanol alkyl ethers and their analogues, chenodeoxycholates, deoxycholates, and mixtures thereof. The micelle-forming compounds may be added simultaneously with or after the addition of alkali metal alkyl sulfates. Mixed micelles are formed with substantially any type of mixture of components, but vigorous mixing is preferred to provide smaller sized micelles.

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

[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 isotonic agent such as glycerin may also be added after the formation of the mixed micelle composition.

[0228] In the delivery of the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, and the dispenser is filled with an ejectant. The ejectant under pressure is in liquid form in the dispenser. The ratio of the components is adjusted so that the aqueous phase and the ejectant phase become one, that is, so that there is one phase. If two phases are present, for example, it is necessary to shake the dispenser before dosing a part of the contents by means of a metering valve. The dosing dose of the pharmaceutical is sprayed from the metering valve in a fine spray form.

[0229] The ejectant 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 concentration of the essential components can be determined by relatively simple experiments. For absorption through the oral cavity, it is often desirable to increase, for example, at least two or three times the dosage for administration by injection or via the gastrointestinal tract.

[0231] B. Nucleic acid lipid particles The iDNA of the present invention, i.e., APOC3 dsRNA, may be completely encapsulated in a lipid formulation to form, for example, SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle containing SPLP. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle containing plasmid DNA encapsulated within a lipid vesicle. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALP and SPLP are extremely useful for systemic application because they have an extended circulating lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLP includes "pSPLP," which contains an encapsulated condensant-nucleic acid complex, as shown in PCT International Publication No. 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, when nucleic acids are present in the nucleic acid-lipid particles of the present invention, they are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent No. 5,976,567; U.S. Patent No. 5,981,501; U.S. Patent No. 6,534,484; U.S. Patent No. 6,586,410; U.S. Patent No. 6,815,432; U.S. Patent Publication No. 2010 / 0324120 and PCT International Publication No. 96 / 40964.

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

[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-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-di Methylaminopropane (DLin-C-DAP), 1,2-Dilinoley oxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoley oxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoley-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoley thio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleyl-2-Linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoley oxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoley-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogues thereof The following may be used: (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-yl4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazanegiyl)didodecane-2-ol (Tech G1), or mixtures thereof. Cationic lipids can constitute approximately 20 mol% to 50 mol%, or even 40 mol%, of the total lipids present in the particles.

[0234] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane may 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 contain 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (mol percent), with a particle size of 63.0 ± 20 nm and a siRNA / lipid ratio of 0.027.

[0236] Ionic / non-cationic lipids include, without limitation, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleyl-phosphatidylethanolamine 4-(N The anionic or neutral lipids may include -maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidy(phosphatidy)ethanolamine (SOPE), cholesterol, or mixtures thereof. If cholesterol is included, the non-cationic lipids may constitute about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0237] The conjugate lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG)-lipids containing, non-limitingly, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, 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 (C8). The amount of conjugate lipids that prevent particle aggregation can be 0 mol% to about 20 mol%, or 2 mol%, of the total lipids present in the particles.

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

[0239] In one embodiment, lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) may be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Each stock solution in ethanol may be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-Ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-Ceramide C16 may then be combined, for example, in a molar ratio of 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., in sodium acetate (pH 5)) such that the final ethanol concentration is approximately 35–45% and the final sodium acetate concentration is approximately 100–300 mM. Lipid-dsRNA nanoparticles are usually formed spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., with a 100 nm cutoff) using a thermobarrel extruder, such as a Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate-buffered saline (PBS) of approximately pH 7, e.g., approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. [ka]

[0240] The LNP01 formulation is described, for example, in the brochure International Publication No. 2008 / 042973, which is incorporated herein by reference.

[0241] Additional exemplary lipid-dsRNA formulations are set forth in the following table.

[0242] [Table 1]

[0243] [Table 2]

[0244] [Table 3]

[0245] DSPC: Distearoyl phosphatidylcholine DPPC: Dipalmitoyl phosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG having an average molecular weight of 2000) PEG-DSG: PEG-distearyl glycerol (C18-PEG, or PEG-C18) (PEG having an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG having an average molecular weight of 2000) Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. WO 2009 / 127060, filed Apr. 15, 2009, which is hereby incorporated by reference herein.

[0246] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366 filed on 29 January 2009; U.S. Provisional Patent Application No. 61 / 156,851 filed on 2 March 2009; U.S. Provisional Patent Application No. [number missing] filed on 10 June 2009; U.S. Provisional Patent Application No. 61 / 228,373 filed on 24 July 2009; U.S. Provisional Patent Application No. 61 / 239,686 filed on 3 September 2009; and International Application PCT / US2010 / 022614 filed on 29 January 2010.

[0247] Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed on June 10, 2010, which is incorporated herein by reference in its entirety.

[0248] Formulations containing ALNY-100 are described, for example, in International Patent Application PCT / US09 / 63933, filed November 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 Patent 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 used in the nucleic acid-lipid particles of the present invention, such as cationic lipids, can be prepared by known organic synthesis techniques, including the methods described in detail in the examples. All substituents are defined below unless otherwise specified.

[0251] "Alkyl" refers to a saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms, in a linear or branched, acyclic or cyclic form. Typical saturated linear alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl; typical saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; and unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl.

[0252] "Alkenyl" refers to the alkyl group defined above, which contains at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative linear and branched alkenyls include ethyleneyl, propyrenyl, 1-butenyl, 2-butenyl, isobutyrenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl.

[0253] "Alkynyl" means any alkyl or alkenyl as defined above, further containing at least one triple bond between adjacent carbon atoms. Typical linear and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0254] "Acyl" refers to any alkyl, alkenyl, or alkynyl group in which the carbon at the bonding site 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] A "heterocycle" means a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocycle that is 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), and this heterocycle includes a bicyclic ring in which any of the above heterocycles is fused to a benzene ring. Heterocycles may be bonded via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxyranil, oxetanyl, tetrahydrofuranil, tetrahydropyranil, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranil, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranil, etc.

[0256] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle" all mean that when substitution occurs, at least one hydrogen atom is substituted by the substituent. In the case of an oxo substituent (=O), two hydrogen atoms are substituted. In this regard, the substituents include oxo, halogen, heterocycle, -CN, -ORx, -NRxRy, -NRxC(=O)Ry, -NRxSO2Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SOnRx and -SOnNRxRy, where n is 0, 1 or 2, Rx and Ry are the same or different, and independently hydrogen, alkyl or heterocycle, and each of the alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORx, heterocycle, -NRxRy, -NRxC(=O)Ry, -NRxSO2Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SOnRx and -SOnNRxRy.

[0257] "Halogen" refers to fluoro, chloro, bromo, and iodine.

[0258] In some embodiments, the method of the present invention may require the use of protecting groups. Methods of protecting groups 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 undesirable reactivity of a functional group. Protecting groups can be added to a functional group to shield its reactivity in 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 undesirable reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0259] Synthesis of Equation A In one embodiment, the nucleic acid-lipid particles of the present invention are of formula A: [ka] The formulation is made using the cationic lipid, 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 together can form an optionally substituted heterocycle. In one embodiment, 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 specified.

[0260] Scheme 1 [ka] Lipid A (wherein R1 and R2 are independently alkyl, alkenyl, or alkynyl, each optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 together can 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. The reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields the lipid of formula A. The lipid of formula A can be converted to the corresponding ammonium salt using the organic salt of formula 5 (wherein X is an anion counterion selected from halogens, hydroxides, phosphates, sulfates, etc.).

[0261] Scheme 2 [ka] Alternatively, the starting materials for ketone 1 may be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 may be purchased or prepared according to methods known to those skilled in the art. The reaction of 6 and 7 yields ketone 1. The conversion of ketone 1 to the corresponding lipid of formula A is shown in Scheme 1.

[0262] MC3 synthesis 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-dimethylaminobutyrate (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 washing with dilute sodium bicarbonate aqueous solution. The organic fraction was dried on anhydrous magnesium sulfate, filtered, and the solvent was removed in a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. The fractions containing the purified product were combined, the solvent was removed, and a colorless oil (0.54 g) was obtained.

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

[0264] 515 synthesis In a two-necked round-bottom flask (1 L), a stirring suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml of anhydrous THF was slowly added to a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF at 0°C under a nitrogen atmosphere. After complete addition, the reaction mixture was warmed to room temperature and then heated under reflux for 4 hours. The progress of the reaction was monitored by TLC. After the completion of the reaction (by TLC), the mixture was cooled to 0°C and quenched by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 hours and then filtered to remove the residue. The residue was thoroughly washed with THF. The filtrate and washing solution were mixed and diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 minutes. Volatile substances were removed under reduced pressure to obtain 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] 516 synthesis In a 250 mL two-necked round-bottom flask, NET3 (37.2 mL, 0.2669 mol) was added to a stirred solution of compound 515 in 100 mL of dry DCM, and the mixture was cooled to 0°C under a nitrogen atmosphere. N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM was slowly added, and the reaction mixture was warmed to room temperature. After the reaction was complete (by TLC for 2-3 hours), the mixture was successively washed with 1N HCl solution (1 × 100 mL) and saturated NaHCO3 solution (1 × 50 mL). Next, the organic layer was dried on anhydrous Na2SO4, and the solvent was evaporated to obtain the crude material, which was purified by silica gel column chromatography to obtain 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] Combination 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 round-bottom flask. N-methylmorpholine N-oxide (7.6 g, 0.06492 mol) was then 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 the reaction was complete (approximately 3 hours), the mixture was quenched by adding solid Na2SO3, and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO3 (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. A mixture of diastereomers was obtained by silica gel column chromatography purification of the crude material, and separated by preparative HPLC. Yield: 6g of crude 517A-peak-1 (white solid), 5.13g (96%). ¹H-NMR (DMSO, 400MHz): 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] 518 synthesis Compound 518 (1.2 g, 41%) was obtained as a colorless oil using the same procedure as described for the synthesis of compound 505. 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 equivalent) in hexane (15 mL) was added dropwise to an ice-cooled solution of LAH in THF (1 M, 2 equivalents). After complete addition, the mixture was heated at 40°C for 0.5 hours and then cooled again on an ice bath. The mixture was carefully hydrolyzed with a saturated aqueous solution of Na₂SO₄, then filtered through Celite and reduced to an oil. Column chromatography yielded pure 519 (1.3 g, 68%), which was obtained as a colorless oil. 13 ¹¹C 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 value 654.6, measured value 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 free of aggregates or precipitates. The particle size and particle size distribution of lipid-nanoparticles can be measured, for example, by light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). The particles should be approximately 20–300 nm in size, e.g., 40–100 nm. The particle size distribution should be unimodal. The total dsRNA concentration and captured percentage in the formulation are estimated using a dye exclusion assay. A sample of formulated dsRNA may be incubated with an RNA-binding dye such as Ribogreen (Molecular Probes) in or out of the presence of a formulation-disintegrating surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the surfactant-containing sample against a standard curve. The captured percentage is determined by subtracting the "free" dsRNA content (measured by signal in the absence of surfactant) from the total dsRNA content. The percentage of captured dsRNA is typically >85%. For SNALP formulations, particle sizes are at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Preferred ranges are typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0270] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, turbidiants, or liquids in water or a non-aqueous medium, capsules, gel capsules, medicine bags, tablets, or small tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are desired. In some embodiments, the oral formulation is administered with one or more permeabilizing surfactants and chelating agents, containing the dsRNA characteristic of the present invention. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, combinations of osmotic enhancers, such as fatty acid / salt combinations with bile acids / salts, are used. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNAs characterizing the present invention can be delivered orally in granular form, including spray-dried particles or those complexed to form micro or nanoparticles.Examples of dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates; cationic gelatin, albumin, starch, acrylates, polyethylene glycol (PEG), and starch; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pullulan, cellulose, and starch. Preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyano(cyano)acrylate), DEAE-methacrylate, DEAE-hexylacrylate Examples include benzoate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations for dsRNA, and such formulations, are described in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

[0271] Compositions and formulations for parenteral, intraparenchymal (into the brain), subarachnoid, intraventricular, or intrahepatic administration include sterile aqueous solutions, which may include buffers, diluents, and other suitable additives, not limited to osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0272] The pharmaceutical compositions of the present invention include, but are not limited to, liquid formulations, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but are not limited to, pre-formed liquid formulations, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases such as hepatocarcinoma, formulations targeting the liver are particularly preferred.

[0273] The pharmaceutical formulations of the present invention, which can conveniently exist in unit dosage forms, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. Generally, the formulations are prepared by homogeneously and intimately associating the active ingredient with a liquid carrier or a micronized solid carrier or both, and then, if necessary, shaping the product.

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

[0275] C. Further Formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. An emulsion is typically a heterogeneous system in which one liquid is dispersed in another liquid, usually in the form of droplets with a diameter greater than 0.1 μm (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and (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). An emulsion is often a two-layer system containing two immiscible liquid phases that are closely mixed and dispersed with each other. Generally, emulsions can be either water in oil (w / o) or oil in water (o / w). When the aqueous phase is finely divided and dispersed as microdroplets in the oil phase of the mass, the resulting composition is called a water in oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as microdroplets in the aqueous phase of the mass, 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 exist as a solution in the aqueous phase, the oil phase, or as a separate phase themselves. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions can be polyemulsions consisting of three or more phases, such as oil-in-water (o / w / o) and water-in-oil (w / o / w) emulsions. Such complex formulations often offer certain advantages not provided by simple two-component emulsions. A polyemulsion in which individual oil droplets of an o / w emulsion surround smaller water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets surrounded by water spheres stabilized in a continuous phase of oil provides an o / w / o emulsion.

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

[0277] Synthetic surfactants, also known as surfactants, have found broad applicability in emulsion formulation and are outlined in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199). Surfactants are typically amphiphilic and contain both hydrophilic and hydrophobic parts. The ratio of hydrophilicity to hydrophobicity in a surfactant is called the hydrophilic / lipophilic balance (HLB), and it is a valuable tool for classifying and selecting surfactants during the preparation of pharmaceutical formulations. Surfactants can be classified into different types based on the properties of their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; and 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. Absorbent bases, such as anhydrous lanolin and hydrophilic petrolatum, incorporate water to form w / o emulsions while retaining their hydrophilic properties to maintain their semi-solid consistency. Micronized solids are used as good emulsifiers, particularly 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 aluminum magnesium 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. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199).

[0280] Hydrophilic colloids include naturally occurring rubbers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), synthetic polymers, cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These disperse in water or swell in water to form a colloidal solution that stabilizes the emulsion by forming a strong interfacial film around droplets of the dispersed phase and by increasing the viscosity of the outer phase.

[0281] Because emulsions often contain numerous components such as carbohydrates, proteins, sterols, and phosphatides that readily support microbial growth, these formulations frequently incorporate preservatives. Commonly used preservatives in formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also typically added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, as well as antioxidant synergists such as citric acid, tartaric acid, and lecithin.

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

[0283] ii. Microemulsion In one embodiment of the present invention, the iRNA and nucleic acid composition is formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and an amphiphilic substance that is a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, a microemulsion is prepared by first dispersing the oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally an alcohol of an intermediate chain length, to form a clear system. Therefore, microemulsions are described as thermodynamically stable, isotropically transparent dispersions consisting of two immiscible liquids stabilized by an interfacial film of surfactant 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 typically prepared using a combination of 3 to 5 components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is water in oil (w / o) or oil in water (o / w) depends on the properties of the oil and surfactant used, as well as the structure of the polar head and hydrocarbon tail of the surfactant molecule and its geometric packing (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

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

[0285] Surfactants used in the preparation of microemulsions include, without limitation, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with auxiliary surfactants. Auxiliary surfactants, typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, penetrate the surfactant film and, as a result, increase interfacial fluidity by forming an irregular film through the resulting void spaces between surfactant molecules. However, microemulsions can be prepared without the use of auxiliary surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, without limitation, water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can not necessarily include materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolated glycerides, saturated polyglycolated C8-C10 glycerides, vegetable oils, and silicone oils.

[0286] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed for improving the oral bioavailability of peptide-containing drugs (see, for example, U.S. Patents 6,191,105, 7,063,860, 7,070,802, and 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as improved drug solubilization, protection of drugs from enzymatic hydrolysis, potentially improved drug absorption due to altered membrane fluidity and permeability by surfactant induction, ease of preparation, ease of oral administration beyond solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Patent Nos. 6,191,105, 7,063,860, 7,070,802, and 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 their components are brought together at ambient temperature. This can be particularly advantageous when formulating heat-sensitive drugs, peptides, or iRNAs. Microemulsions are also effective for the efficient delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to promote increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improved local cellular uptake of iRNAs and nucleic acids.

[0287] The microemulsion of the present invention may also contain 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 iRNA and nucleic acids of the present invention. Permeation enhancers used in the microemulsion of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0288] iii. Particulates The RNAi agent of the present invention may be incorporated into particles, such as microparticles. Microparticles may be produced by spray drying, but may also be produced by other methods including freeze-drying, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0289] iv. Penetration enhancers In one embodiment, the present invention utilizes various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been found that non-lipophilic drugs can also cross cell membranes if the membrane being crossed is treated with a penetration enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the penetration of lipophilic drugs.

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

[0291] Surfactants (or "surface-activating agents") are chemical substances that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of iRNA through mucous membranes. In addition to bile salts and fatty acids, examples of these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluoro compound 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-monoleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, and their C 1~20 Examples include alkyl esters (e.g., methyl, isopropyl, and t-butyl), as well as their monoglycerides and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, for example, Touitou, E., et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0293] The physiological roles of bile include promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as osmotic enhancers. Therefore, the term “bile salts” includes any of the natural components of bile and any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glutolic acid (sodium glutolate), 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-dihydrofusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee). et al.,Critical Reviews in Therapeutic Drug Carrier Systems,1991,page 92;Swinyard,Chapter 39 In:Remington's Pharmaceutical Sciences,18th Ed.,Gennaro,ed.,Mack Publishing Co.,Easton,Pa.,1990,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] A chelating agent used in connection with the present invention can be defined as a compound that removes metal ions from a solution by forming a complex with them, resulting in improved absorption of iRNA through the mucous membrane. Regarding the use of chelating agents as penetration enhancers in the present invention, since most characterized DNA nucleases require divalent metal ions for catalytic activity and are therefore inhibited by chelating agents, chelating agents have the additional advantage of also acting as DNase inhibitors (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 (enamine) (see, for example, Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0295] As used herein, non-chelating, non-surfactant osmotic enhancers may be defined as compounds that exhibit slight activity as chelating agents or surfactants, but nevertheless promote the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of this type of osmotic enhancer include, 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 drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0296] Agents that promote iRNA uptake at the cellular level may also be added to the pharmaceutical compositions and other compositions of the present invention. For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188 by Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (International Publication No. 97 / 30731 of the PCT application by Lollo et al.) are also known to promote dsRNA uptake by cells. Examples of commercially available transfection reagents include, in particular, Lipofectamine(trademark) (Invitrogen; Carlsbad, CA), Lipofectamine 2000(trademark) (Invitrogen; Carlsbad, CA), 293fectin(trademark) (Invitrogen; Carlsbad, CA), Cellfectin(trademark) (Invitrogen; Carlsbad, CA), DMRIE-C(trademark) (Invitrogen; Carlsbad, CA), FreeStyle(trademark)MAX(Invitrogen; Carlsbad, CA), and Lipofectamine(trademark) 2000. 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 (trademark) Transfection Reagent (Promega; Madison, WI), Tfx (trademark)-20 Reagent (Promega; Madison, WI), Tfx (trademark)-50 Reagent (Promega; Madison, WI), DreamFect (trademark) (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass; aD1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM) / LipoGen(TM) (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER(TM) transfection Reagent (Genlantis; San Examples include Diego, CA, USA; RiboFect (Bioline; Taunton, MA, USA); PlasFect (Bioline; Taunton, MA, USA); UniFECTOR (B-Bridge International; Mountain View, CA, USA); SureFECTOR (B-Bridge International; Mountain View, CA, USA); or HiFect (trademark) (B-Bridge International, Mountain View, CA, USA).

[0297] The penetration of administered nucleic acids can be enhanced using other agents, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azon, and terpenes such as limonene and menthone.

[0298] v. Carrier Certain compositions of the present invention also incorporate a carrier compound in the formulation. As used herein, “carrier compound” or “carrier” may be inert (i.e., lacking biological activity), but may refer to a nucleic acid or analogue recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids, for example, by degrading biologically active nucleic acids or facilitating the removal of biologically active nucleic acids from circulation. Co-administration of nucleic acids and carrier compounds, typically with an excess of the latter, can substantially reduce the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory reservoirs, possibly due to competition between the carrier compound and nucleic acid for a common receptor. For example, partial recovery of phosphorothioates in liver tissue may be reduced when co-administered with polyinosinate, dextran sulfate, polycytidic acid, or 4-acetamide-4'isothiocyanostilbene-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 carrier compounds, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected to provide the desired bulk, consistency, etc., when combined with nucleic acids 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 (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); tablet disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0300] Suitable organic or inorganic excipients that do not react harmfully with nucleic acids and are pharmaceutically acceptable for parenteral administration may also be used in the formulation of the compositions of the present invention. Suitable pharmaceutically 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, and polyvinylpyrrolidone.

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

[0302] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0303] vii. Other components The compositions of the present invention may further contain other auxiliary components conventionally found in pharmaceutical compositions, at levels of use established in the art. Therefore, for example, the compositions may contain further, suitable, pharmaceutically active materials such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents, or further materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, these materials, when added, must not excessively 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 acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0304] The aqueous turbidity solution may contain substances that increase the viscosity of the turbidity solution, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The turbidity solution may also contain stabilizers.

[0305] In some embodiments, the pharmaceutical compositions covered by the present invention comprise (a) one or more iRNA compounds and (b) one or more agents that function by a non-RNAi mechanism and are useful for treating lipid metabolism disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-lipid agents, antiviral agents, and / or anti-fibrotic agents. Furthermore, other substances commonly used to protect the liver, such as silymarin, may also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include protease inhibitors such as terbivudine, entecavir, and telaprevir, as well as other agents disclosed, for example, in U.S. Patent Application Publications 2005 / 0148548, 2004 / 0167116, and 2003 / 0144217 by Tung et al.; and U.S. Patent Application Publication 2004 / 0127488 by Hale et al.

[0306] The toxicity and therapeutic effects of such compounds are, for example, LD 50 (Lethal dose for 50% of the population) and ED 50 The dose effective for treatment in 50% of the population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic effect and treatment effect is the treatment index, LD50. 50 / ED 50 It can be expressed as a ratio. Compounds exhibiting a high treatment index are preferred.

[0307] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for human use. The dosages of the compositions discussed herein are generally administered with little to no toxicity. 50The blood concentration range includes [the specified concentration]. The dose may vary within this range depending on the dosage form and route of administration used. For any compound used in the methods addressed in this invention, the therapeutically effective dose can be initially inferred from a cell culture assay. The dose can be measured in cell cultures and formulated to achieve in an animal model the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence, including the IC50 (i.e., the concentration of the test compound that achieves half of the maximum inhibition of symptoms) (e.g., to achieve a decrease in polypeptide concentration). Using such information, a useful dose in humans can be determined more accurately. Plasma levels can be measured, for example, by high-performance liquid chromatography.

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

[0309] VI. Method of the present 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 cells. The method comprises the steps of contacting cells with the dsRNA of the present invention and maintaining the cells for a time sufficient to obtain degradation of the mRNA transcript of the ANGPTL3 gene, thereby inhibiting the expression of the ANGPTL3 gene in cells. The reduction in gene expression can be evaluated by any method known in the art. For example, the reduction in ANGPTL3 expression may be measured by methods routine to those skilled in the art, such as measuring the mRNA expression level of ANGPTL3 using Northern blotting or qRT-PCR; or by measuring the protein level of ANGPTL3 using methods routine to those skilled in the art, such as Western blotting or immunological techniques. The reduction in ANGPTL3 expression may also be evaluated indirectly by measuring the reduction in the biological activity of ANGPTL3, such as the reduction in serum lipids, triglycerides, cholesterol and / or free fatty acid levels.

[0310] In the method of the present invention, cells may be contacted in vitro or in vivo, that is, cells may be within the object.

[0311] Cells suitable for treatment using the method of the present invention may be any cells expressing the ANGPTL3 gene. Suitable cells for use in the method of the present invention may be mammalian cells, such as primate cells (human or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (for example, duck cells or goose cells), or whale cells. In one embodiment, the cells are human cells, such as human liver cells.

[0312] ANGPTL3 expression is present in cells at a rate of at least approximately 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, 5 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 approximately 100% inhibition.

[0313] The in vivo method of the present invention may include administering a composition containing iRNA to a target, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of the 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 can be administered by any means known in the art, including, but not limited to, oral, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and subarachnoid), intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), nasal, rectal, and topical (including intrabuccal and sublingual) parenteral routes. 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 allows for consistent release of iRNA over a long period of time. Therefore, depot injection can reduce the frequency of administration required to obtain the desired effect, such as the desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. Depot injection can also provide more consistent serum concentrations. Depot injection may include subcutaneous or intramuscular injection. In preferred embodiments, the depot injection is subcutaneous.

[0315] In some embodiments, administration is by pump. The pump may be an external pump or a surgically implanted pump. In a given embodiment, 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, intra-arterial, 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 iRNA to the liver.

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

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

[0318] The present invention further provides a method for treating subjects requiring treatment. The treatment method of the present invention comprises administering the iRNA of the present invention to a subject, for example, a subject who may benefit from a reduction and / or inhibition of ANGPTL3 expression, in a therapeutically effective amount of iRNA targeting the ANGPTL3 gene or a pharmaceutical composition containing iRNA targeting the ANGPTL3 gene.

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

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

[0321] Subjects who may benefit from reduced and / or inhibited ANGPTL3 gene expression are those suffering from lipid metabolism disorders, such as hereditary or acquired lipid metabolism disorders. In one embodiment, subjects suffering from lipid metabolism disorders are also suffering from hyperlipidemia. In another embodiment, subjects suffering from lipid metabolism disorders are also suffering from hypertriglyceridemia. Treatment for subjects who may benefit from reduced and / or inhibited ANGPTL3 gene expression includes therapeutic treatment (e.g., subjects suffering from xanthelasma) and prophylactic treatment (e.g., subjects not suffering from xanthelasma, or subjects at risk of developing xanthelasma).

[0322] The present invention further provides methods for using iRNAs or pharmaceutically active compositions thereof for treating subjects who may benefit from reduced and / or inhibition of ANGPTL3 expression, for example, subjects suffering from lipid metabolism disorders, in combination with other pharmaceuticals and / or other treatment methods, for example, those currently used to treat these disorders, such as known pharmaceuticals and / or known treatment methods. For example, in certain embodiments, ANGPTL3-targeting iRNAs are administered in combination with agents useful for treating lipid metabolism disorders, for example, as described elsewhere herein. For example, further agents suitable for treating subjects who may benefit from reduced ANGPTL3 expression, for example, subjects suffering from lipid metabolism disorders, may include agents that reduce one or more serum lipids. Non-limiting examples of such agents may include HMG-CoA reductase inhibitors, such as cholesterol synthesis inhibitors like 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 activator (SCAP) activators; microsomal triglyceride transfer protein (MTP) inhibitors; ApoE-related peptides; and treatment antibodies against ANGPTL3. Further treatment agents may include agents that increase high-density lipoprotein (HDL), such as cholesteryl ester transfer protein (CETP) inhibitors. Furthermore, further treatment agents may include nutritional supplements, such as fish oil.iRNA and further treatment agents may be administered simultaneously and / or in the same combination, for example, parenterally, or the further treatment agents may be administered as part of a separate composition, or at a different time, 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 the expression of a target ANGPTL3 gene is reduced for, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the expression of the target ANGPTL3 gene is reduced for, for example, at least about 2, 3, 4 days or longer, for example, for an extended duration of about 1 week, 2 weeks, 3 weeks, or 4 weeks or longer.

[0324] Preferably, the iRNAs useful in the methods and compositions described 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 may be prepared and carried out as described herein.

[0325] Administration of dsRNA according to the method of the present invention may reduce the severity, signs, symptoms, and / or markers of such disease or disorder in patients with lipid metabolism disorders. In this context, “reduction” means a statistically significant reduction in those levels. The reduction may 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 a treatment or prevention of disease can be evaluated by measuring, for example, disease progression, disease remission, symptom severity, pain reduction, quality of life, the dosage of medication required to sustain the treatment effect, levels of disease markers, or any other measurable parameter appropriate to a given disease being treated or targeted for prevention. Monitoring the effectiveness of a treatment or prevention by measuring any one of these parameters, or any combination of parameters, is within the scope of the skills of those skilled in the art. For example, the effectiveness of a treatment for lipid metabolism disorders can be evaluated, for example, by periodic monitoring of one or more serum lipid levels. A comparison of initial and subsequent readings provides the physician with an indicator of whether the treatment is effective. Monitoring the effectiveness of a treatment or prevention by measuring any one of these variables, or any combination of variables, is well within the scope of the skills of those skilled in the art. "Effective against" lipid metabolism disorders in association with the administration of iRNA or a pharmaceutical composition thereof targeting ANGPTL3 means that administration in a clinically appropriate manner results in a statistically significant proportion of patients with beneficial effects such as improvement of symptoms, cure, reduction of disease, extension of lifespan, improvement of quality of life, or other effects generally recognized as favorable by physicians familiar with the treatment of lipid metabolism disorders and related causes.

[0327] The effectiveness of a treatment or preventive measure is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening or the expected symptoms do not occur. For 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 effectiveness of an iRNA drug, or a given formulation of such drug, can also be determined using an experimental animal model for a given disease, as is known in the art. When using an experimental animal model, the effectiveness of the treatment is demonstrated when a statistically significant decrease is observed in a marker or symptom.

[0328] Alternatively, efficacy can be measured by a reduction in disease severity, as determined by those skilled in the art based on clinically acceptable disease severity assessment measures, such as the Child-Pugh score (or occasionally the Child-Turcotte-Pugh score). Any positive change resulting in, for example, a reduction in disease severity, as measured using an appropriate measure, represents adequate treatment with the iRNA or iRNA preparations described herein.

[0329] The target group included approximately 0.01 mg / kg to approximately 5 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.05 mg / kg to approximately 5 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 0.2 mg / kg to approximately 5 mg / kg, approximately 0.2 mg / kg to approximately 10 mg / kg, and approximately 0.3 mg / kg to approximately 5 mg / kg. Approximately 0.3 mg / kg to approximately 10 mg / kg, approximately 0.4 mg / kg to approximately 5 mg / kg, approximately 0.4 mg / kg to approximately 10 mg / kg, approximately 0.5 mg / kg to approximately 5 mg / kg, approximately 0.5 mg / 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 Therapeutic doses of dsRNA such as 10 mg / kg, approximately 6 mg / kg to approximately 10 mg / kg, approximately 6.5 mg / kg to approximately 10 mg / kg, approximately 7 mg / kg to approximately 10 mg / kg, approximately 7.5 mg / kg to approximately 10 mg / kg, approximately 8 mg / kg to approximately 10 mg / kg, approximately 8.5 mg / kg to approximately 10 mg / kg, approximately 9 mg / kg to approximately 10 mg / kg, or approximately 9.5 mg / kg to approximately 10 mg / kg may be administered. Values ​​up to the cited values ​​and intermediate ranges are intended to be part of the present invention.

[0330] For example, dsRNA is approximately 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, It may be administered in doses of 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 between the cited values ​​are intended to be part of the present invention.

[0331] In another embodiment, for example, if the composition of the present invention contains the dsRNA and N-acetylgalactosamine described herein, the target ranges are approximately 0.1 to approximately 50 mg / kg, approximately 0.25 to approximately 50 mg / kg, approximately 0.5 to approximately 50 mg / kg, approximately 0.75 to approximately 50 mg / kg, approximately 1 to approximately 50 mg / kg, approximately 1.5 to approximately 50 mg / kg, approximately 2 to approximately 50 mg / kg, approximately 2.5 to approximately 50 mg / kg, approximately 3 to approximately 50 mg / kg, approximately 3.5 to approximately 50 mg / kg, approximately 4 to approximately 50 mg / kg, approximately 4.5 to approximately 50 mg / kg, approximately 5 to approximately 50 mg / kg, and approximately 7.5 to approximately 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 doses of dsRNA may be administered in amounts such as 0.1 to approximately 20 mg / kg, approximately 0.25 to approximately 20 mg / kg, approximately 0.5 to approximately 20 mg / kg, approximately 0.75 to approximately 20 mg / kg, approximately 1 to approximately 20 mg / kg, approximately 1.5 to approximately 20 mg / kb, approximately 2 to approximately 20 mg / kg, approximately 2.5 to approximately 20 mg / kg, approximately 3 to approximately 20 mg / kg, approximately 3.5 to approximately 20 mg / kg, approximately 4 to approximately 20 mg / kg, approximately 4.5 to approximately 20 mg / kg, approximately 5 to approximately 20 mg / kg, approximately 7.5 to approximately 20 mg / kg, approximately 10 to approximately 20 mg / kg, or approximately 15 to approximately 20 mg / kg. The values ​​up to and between the cited values ​​are intended to be part of the present invention.

[0332] For example, the target values ​​are approximately 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 doses 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 approximately 50 mg / kg may be administered. Values ​​up to and between the cited values ​​are intended to be part of the present invention.

[0333] 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 approximately 25 minutes. Administration may be repeated regularly, for example, every other week (i.e., every two weeks) for one month, two months, three months, four months, or longer. After the initial treatment plan, treatment may be administered less frequently. For example, after administration every other week for three months, administration may be repeated once a month for six months, or for one year, or longer. iRNA administration reduces ANGPTL3 levels in cells, tissues, blood, urine, or other compartments of the patient by at least approximately 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 , 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 can be reduced by at least about 99%, or more.

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

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

[0336] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Similar or equivalent methods and materials may be used in the practice or testing of the iRNAs and methods characterizing this invention, although preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to be limiting. [Examples]

[0337] Example 1: iRNA synthesis Reagent supply source Unless otherwise specified herein, reagent sources may be obtained from any supplier in molecular biology, meeting the quality / purity standards required for molecular biology applications.

[0338] Transfer To identify siRNAs targeting cynomolgus monkey (Macaca fascicularis; hereafter "cyno") ANGPTL3 transcripts produced by sequencing human ANGPTL3 transcripts annotated in the NCBI Gene database (http: / / www.ncbi.nlm.nih.gov / gene / ) and cDNA prepared from liver RNA, we designed siRNAs. 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 design: human - NM_014495.2 (SEQ ID NO: 1); mouse - NM_013913.3 (SEQ ID NO: 2). All siRNA double-stranded transcripts that shared 100% identity with the listed human and cyno transcripts were designed. The subset of siRNA double-stranded transcripts described below also shared 100% identity with the mouse (Mus musculus) ANGPTL3 transcript found in the NCBI Gene database.

[0340] siRNA design, specificity, and efficacy prediction The predicted specificity of all possible 19mers was predicted from each sequence. Next, candidate 19mers without repeats longer than 7 nucleotides were selected. Then, these 977 candidate human / cyno siRNAs, and 38 subsets that also matched mouse ("human / cyno / mouse candidate siRNAs"), were used for 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 performed with the Python script "BruteForce.py". The script then analyzes the transcript-oligo alignment to generate a score based on the location and number of mismatches between the siRNA and any possible "off-target" transcripts. The off-target score is weighted to highlight differences in the siRNA "seed" region, located at positions 2-9 from the 5' end of the molecule. A mismatch score was assigned to each oligo-transcript pair by summing the individual mismatch scores obtained through brute-force search; mismatches at positions 2-9 were considered 2.8, mismatches at cleavage sites 10-11 were considered 1.2, and mismatches in region 12-19 were considered 1.0. Further off-target prediction was performed by comparing the frequencies of heptamers and octamers derived from three different seed-directed hexamers for each oligo. Two heptamers and one octamer were generated using hexamers from positions 2-7 relative to the 5' start point. "Heptamer 1" was generated by adding 3'A to the hexamer; "Heptamer 2" was generated by adding 5'A to the hexamer; and an octamer was generated by adding A to both the 5' and 3' ends of the hexamer. The frequencies of octamers and heptamers in the human 3'UTRome (defined as a subsequence of the transcriptome from the NCBI Refseq database, where the end of the code region "CDS" is clearly defined) were precalculated. The octamer frequencies were standardized relative to the heptamer frequencies using the median of the range of octamer frequencies.Next, the "mirSeedScore" was calculated by summing ((3 × standardized octamer value) + (2 × heptamer 2 value) + (1 × heptamer 1 value)).

[0341] Both siRNA strands were assigned to specificity categories according to their calculated scores: scores greater than 3 were considered highly specific, scores equal to 3 were considered specific, and scores between 2.2 and 2.8 were considered moderately specific. Sorting was performed based on the specificity of the antisense strands. Next, double-stranded sequences were selected from human / cyno sets containing antisense oligos without miRNA seed matches (score of 3 or greater, total GC content less than 65%, no GC at the 1st position, 4 or more Us or As in the seed region, and GC at the 19th position). Double-stranded sequences were also selected from human / cyno / mouse sets containing antisense oligos with a score of 2 or greater, total GC content less than 65%, and no GC at the 1st position.

[0342] Selection of siRNA sequences A total of 47 sense and 47 antisense siRNAs were synthesized from human / cyno siRNA oligos and formed into double strands. A total of 15 sense and 15 antisense siRNAs were synthesized from human / cyno / mouse siRNAs and formed into double strands.

[0343] Synthesis of ANGPTL3 sequences The ANGPTL3 sequence was synthesized in either 1 or 0.2 μmol in a MerMade 192 synthesizer. Single-stranded sequences 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 prepared for use in free-uptake screening assays. In these designs, the GalNAc moiety was positioned at the 3' end of the sense strand. The antisense sequence was 23 nucleotides long and contained 2'F and 2'O-methyl chemical modifications with two phosphorothioate bonds at the 3' end.

[0344] In the single-stranded and double-stranded 21-mers, the "endolight" chemistry was applied, as detailed below. All pyrimidines (cytosine and uridine) in the sense strand were modified with 2'-O-methyl nucleotides (2'O-methyl C and 2'O-methyl U). In the antisense chain, the pyrimidines adjacent to the ribo-A nucleoside (towards the 5' position) were substituted with their corresponding 2'-O-methyl nucleosides. • Two dTsdT extensions were introduced to the 3' ends 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-stranded sequences were synthesized. Syntheses were performed on a 1 μmol scale, using GalNAc-modified CPG support for the sense strand and universal support-modified CPG for the antisense sequence. A sequence motif named TOFFEE was applied, where the sense strand contained 3-nucleotide 2'F modification motifs at positions 9, 10, and 11, and the antisense sequence contained 2'O methyl-modified motifs at positions 11, 12, and 13.

[0346] Synthesis, cleavage, and deprotection The ANGPTL3 sequence was synthesized using solid-phase supported oligonucleotide synthesis via phosphoramidite chemistry. For the 21-mer endright sequence, deoxythymidine CPG was used as the solid support, while for the GalNAc conjugate, a GalNAc solid support was used for the sense chain and a general-purpose CPG for the antisense chain.

[0347] The above sequences were synthesized in 96-well plates at a scale of either 1 or 0.2 μm. An amidite solution was prepared at a concentration of 0.1 M, and ethylthiotetrazole (0.6 M in acetonitrile) was used as the activator.

[0348] The synthesized sequences were cleaved and deprotected in a 96-well plate using methylamine in the first step and a fluoride reagent in the second step. For sequences containing GalNAc and 2'F nucleosides, the deprotection conditions were modified. After cleavage and deprotection, the sequences 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 chromatography was used for quantification, and selected sets of samples were analyzed by IEX chromatography to measure purity.

[0349] Purification, desalting, and annealing The ANGPTL3 sequence was precipitated and purified using a Sephadex column in an AKTA Purifier system. ANGPTL3 was tested at ambient temperature. Sample injection and collection were performed in a 96-well plate with wells of 1.8 mL depth. A single peak corresponding to the full-length sequence was collected in the eluate. The desalted ANGPTL3 sequence was eluted at a concentration (A 260 The siRNA was analyzed for UV radiation and purity (by ion-exchange HPLC). Next, complementary single strands were combined in a 1:1 stoichiometric ratio to form siRNA double strands.

[0350] Example 2. In vitro screening Cell culture and transfection Hep3B cells (ATCC, Manassas, VA) were grown in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) under a 5% CO2 atmosphere at 37°C until near confluence, and then released from the plate by trypsin treatment. Transfection was performed by adding 14.8 μl of Opti-MEM + 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA, cat#13778-150) per well to 5 μl of siRNA double-stranded cells per well in a 96-well plate, and incubated at room temperature for 15 minutes. Subsequently, antibiotic-free cells were subjected to ~2 × 10⁶ exposure. 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 final double-strand concentrations of 10 nM and 0.1 nM, and dose-response experiments were performed at final double-strand concentrations of 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM.

[0351] Free transfection Each 5 μl of GalNac conjugated siRNA in PBS was resuspended in 95 μl of In Vitro Gro CP medium (In Vitro Technologies-Celsis, Baltimore, MD) in each well of a 96-well plate, with 4 × 10⁶ samples being collected. 4 The siRNA was combined with freshly thawed, cryopreserved cynomolgus monkey liver cells. The mixture was incubated in a 5% CO2 atmosphere at 37°C for approximately 24 hours. For efficacy free uptake assays, the 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 the DYNABEADS mRNA isolation kit (Invitrogen, part#:610-12): Cells were harvested, lysed in 150 μl of lysis / binding buffer, and mixed for 5 minutes at 850 rpm using an Eppendorf thermomixer (the mixing rate was consistent throughout the process). 10 microliters of magnetic particles and 80 μl of the lysis / binding buffer mixture were added to a 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 removing the supernatant, the lysed cells were added to the remaining particles and mixed for 5 minutes. After removing 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. Next, the particles were washed with 150 μl of wash buffer B, captured, and the supernatant was removed. Then the particles were 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 at 70°C for 5 minutes. The particles were captured on a magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96-well plate.

[0353] cDNA synthesis using the ABI high-volume cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, Cat#4368813): A master mix consisting of 2 μl of 10X buffer, 0.8 μl of 25X dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNAe inhibitor, and 3.2 μl of H2O was added to 10 μl of total RNA per reaction. cDNA was generated using a Bio-RadC-1000 or S-1000 thermocycler (Hercules, CA) via the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and then held at 4°C.

[0354] Real-time PCR: 2 μl of cDNA was added to a master mix containing 0.5 μl of GAPDHTaqMan 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 using the ΔΔCt(RQ) assay in an ABI7900HT Real Time PCR system (Applied Biosystems). Unless otherwise noted in the summary table, each double-stranded DNA was tested in two independent transfections, and each transfection was assayed twice.

[0355] To calculate the relative doubling change, real-time data was analyzed using the ΔΔCt method and normalized to assays performed using cells transfected with 10 nM AD-1955 or pseudo-transfected cells. The IC50 was calculated using a 4-parameter fitted model with XLFit and normalized over the same dose range or to the lowest dose of AD-1955 transfected cells or untreated cells. The AD-1955 sequence used as a negative control targeted luciferase and had the following sequences: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14); antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 15).

[0356] Survival rate screening Cell viability was measured on days 3 and 6 in HeLa and Hep3B cells after transfection with siRNAs of 10, 1, 0.5, 0.1, and 0.05 nM. Cells were cultured on plates at a density of 10,000 cells per well in a 96-well plate. Each siRNA was assayed three times, and the data were averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for decreased viability, while AD-1955 and mock transfected cells were included as negative controls. PLK1 and AD-19200 resulted in dose-dependent decreases in viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of a 96-well plate at 3 or 6 days and incubated at 37°C for 2 hours. The plates were then read at 560 Ex / 590 Em using a molecular device spectrophotometer. Survival rate was expressed as the mean value in units of light, derived from the ± standard deviation of three repeated transfections. Relative survival rate was assessed by first averaging the three repeated transfections and then standardizing the simulated transfected cells. Data are expressed as the percentage of viable cells.

[0357] Table 1: Abbreviations for nucleotide monomers used to describe nucleic acid sequences. When these monomers are present in oligonucleotides, it can be understood that they are linked 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 conducted using the modified oligonucleotide double strands listed in Table 3. The sequence of AD-15838.2 is identical to that of AD-15838.1. siRNA double strands were delivered using LNPs.

[0368] [Table 13]

[0369] [Table 14]

[0370] [Table 15]

[0371] Table 5. Dose-response screening results for ANGPTL3 dsRNA sequences The experiments were conducted using the modified oligonucleotide double strands listed in Table 3. The sequence of AD-15838.2 is identical to the sequence of AD-15838.1.

[0372] [Table 16]

[0373] Table 6. Results of cell viability screening using modified ANGPTL3 dsRNA sequences Experiments were conducted using the modified oligonucleotide double strands listed in Table 3. The sequence of AD-15838.2 is identical to the sequence of AD-15838.1. Survival rate data are expressed as survival rate % 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 conjugate 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 conjugate 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 using ANGPTL3 GalNac conjugate dsRNA. Modified siRNA was tested by transfection in Hep3b cells and by free uptake in primary cynomolgus monkey (PCH) cells at the above doses.

[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 conjugate dsRNA sequences. A subset of active siRNAs obtained from single-dose screening (see data in Table 11) was tested in dose-response experiments by free uptake in PCH cells. These active siRNA subsets were also tested in dose-response experiments 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 screening.

[0441] [Table 79]

[0442] [Table 80]

[0443] Table 15. IDs of double-stranded pairs synthesized and tested in both unconjugated and GalNac conjugated forms. These double strands 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 the dsRNA sequence of the present invention. 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). The dsRNA sequence used as a negative control was luciferase conjugate. The compound was AD-48399B1 ("Luc", sense sequence: CfaCfuUfaCfgCfuGfaGfuAfcUfuCfgAfL96 (SEQ ID NO: 1728), antisense sequence: uCfgAfaGfuAfcUfcAfgCfgUfaAfgUfgsAfsu (SEQ ID NO: 1729)). GalNal conjugate AD-1955, containing 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 of Luc, or 5 or 20 mg / kg of ANG once daily for 5 days; ob / ob mice were administered 20 mg / kg of Luc-formulated NPL or 20 mg / kg of ANG once daily for 5 days. All test substances were administered by subcutaneous injection according to the procedure shown in Figure 1. Specifically, the test substances were administered once daily for 5 consecutive days (days 0, 1, 2, 3, and 4), and blood samples were collected 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 expression of the ANGPTL3 protein was measured using an ELISA assay with collected blood samples. In addition, 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 in Figure 2 shows the levels 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 in Figure 2 shows the levels of mANGPTL3 protein measured in ob / ob mice after administration of 20 mg / kg of control or ANG. The data show that in both wild-type (WT) and ob / ob mice, administration of ANG reduced the levels of mANGPTL3 protein compared to the control.

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

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

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

[0456] Example 4 Test substance The effect of a single dose of the dsRNA sequence of the present invention on levels of ANGPTL3 protein 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 sequence was examined in human PCS transgenic mice characterized by liver-specific expression of the full-length human PCSK9 gene. Human PCS transgenic mice were administered either AD-52981 or PBS via a single subcutaneous injection. The mice were divided into four groups, each consisting of two males and two females. Each group received either PBS injection or AD-52981 at 5 mg / kg, 20 mg / kg, or 60 mg / kg. Blood samples were collected 1 day before administration, 0 days before administration, and 72 hours after administration. ANGPTL3 protein levels were measured by ELISA and compared to levels 1 day before administration and 0 days before administration.

[0458] result Figure 7 shows the levels of mouse ANGPTL3 protein (mANGPTL3) measured in human PCS transgenic mice. The data shown are expressed in comparison to PBS controls and represent the mean of two males and two females in each group. Error bars represent the standard deviation. The data show that 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 to less than one-fifth (see Figure 7).

[0459] array Sequence ID 1 >gi|41327750|ref|NM_014495.2|Human (Homo sapiens) angiopoietin-like 3 (ANGPTL3), mRNA

[0460] Sequence ID 2 >gi|297278846|ref|XM_001086114.2|PREDICTED: Rhesus macaque (Macaca mulatta) angiopoietin-like 3 (ANGPTL3), mRNA

[0461] Sequence ID 3 >gi|142388354|ref|NM_013913.3|House mouse (Mus musculus) angiopoietin-like 3 (Angptl3), mRNA

[0462] Sequence ID 4 >gi|68163568|ref|NM_001025065.1|Norway rat (Rattus norvegicus) angiopoietin-like 3 (Angptl3), mRNA

[0463] Sequence ID 5 is the inverse complementary sequence of Sequence ID 1.

[0464] Sequence ID 6 is the inverse complementary sequence of Sequence ID 2.

[0465] Sequence ID 7 is the inverse complementary sequence of Sequence ID 3.

[0466] Sequence ID 8 is the inverse complementary sequence of Sequence ID 4.

[0467] Sequence ID 9 Cynomolgus monkey (Macaca fascicularis) angiopoietin-like 3 (Angptl3), mRNA

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3), wherein the dsRNA includes a sense strand and an antisense strand that form a double-stranded region. The sense strand and antisense strand are independently 19 to 23 nucleotides long. The antisense strand comprises at least 17 consecutive nucleotides derived from the nucleotide sequence 5'-AAAAAGACUGAUCAAAUAUGUUG-3' of SEQ ID NO: 472, All nucleotides in the sense strand and all nucleotides in the antisense strand include nucleotide modifications selected from the group consisting of 2'-O-methyl nucleotide modifications, 2'-fluoronucleotide modifications, and nonbasic nucleotide modifications, A ligand containing an N-acetylgalactosamine derivative is conjugated to the sense strand of the dsRNA. dsRNA, or a salt thereof.

2. The dsRNA according to claim 1, or a salt thereof, wherein each chain is independently 21 to 23 nucleotides long.

3. The dsRNA according to claim 1 or 2, or a salt thereof, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

4. A dsRNA according to any one of claims 1 to 3, comprising at least one phosphorothioate or methylphosphonate internucleotide bond, or a salt thereof.

5. A dsRNA according to any one of claims 1 to 4, or a salt thereof, wherein an N-acetylgalactosamine derivative is conjugated to the dsRNA via a divalent or trivalent branched linker.

6. N-acetylgalactosamine derivatives conjugated via trivalent branched linkers, Formula II: 【Chemistry 1】 Formula IX: 【Chemistry 2】 Formula X: 【Transformation 3】 and Equation XIII: 【Chemistry 4】 A dsRNA according to claim 5, or a salt thereof, selected from the group consisting of the following.

7. A pharmaceutical composition for inhibiting the expression of the ANGPTL3 gene, comprising the dsRNA or a salt thereof as described in any one of claims 1 to 6.

8. An in vitro method for inhibiting the expression of ANGPTL3 in cells, a) Contacting the cells with the dsRNA or a salt thereof according to any one of claims 1 to 6; and b) Maintain the cells generated in step (a) for a sufficient amount of time to obtain degradation of the mRNA transcript of the ANGPTL3 gene, thereby inhibiting the expression of the ANGPTL3 gene within the cells. A method that includes this.

9. A pharmaceutical composition for treating a subject suffering from a disorder that can benefit from reduced expression of ANGPTL3, comprising dsRNA or a salt thereof as described in any one of claims 1 to 6.

10. The pharmaceutical composition according to claim 9, wherein the disorder is a lipid metabolism disorder.

11. The pharmaceutical composition according to claim 10, wherein the disorder is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, atherosclerosis, diabetes mellitus, cardiovascular disease, and coronary artery disease.

12. A pharmaceutical composition according to any one of claims 9 to 11, further comprising a further treatment agent.

13. The pharmaceutical composition according to claim 12, wherein the further treatment agent is a statin.

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