NZ620151B2 - Angiopoietin-like 3 (angptl3) irna compostions and methods of use thereof - Google Patents

Abstract

Disclosed is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3 (Angiopoietin-like 3), wherein said dsRNA comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5; wherein the sequences are as defined in the specification. the nucleotide sequence of SEQ ID NO:1 and said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5; wherein the sequences are as defined in the specification.

Description

ANGIOPOIETIN-LIKE 3 (ANGPTL3) iRNA COMPOSITIONS AND METHODS OF USE THEREOF ‘ Related Applications This ation claims priority to US. Provisional ation No. 61/499,620, filed on June 21, 2011, and to US. Provisional Application No, 61/638,288, filed on.

April 25, 2012, the entire contents of each of which are hereby incorporated herein by reference.

Sequence Listing The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, d on July 1 1, 2012, is named 12l30100.txt and is 444,346 bytes in. size.

Background of the Invention Angiopoietin-like 3 (ANGPTL3) is a member ofthe angiopoietin-like family of ed factors that tes lipid metabolism and that is predominantly expressed in the liver (Koishi, R; et al., (2002) Nat. Genet. 30(2):]51-157). ANGPTL3 dually inhibits the catalytic activities of lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and of endothelial lipase (EL), which hydrolyzes high 4 density lipoprotein (HDL) phospholipids. In hypolipidemic, yet obese, KK/Snk mice, a reduction in ANGPTL3 expression has a protective effect against hyperlipidemia and artherosclerosis by promoting the nce of triglycerides (Ando et al., (2003) J. Lipid Res., 442121641223). Human 3 plasma concentrations positively correlate with plasma HDL terol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).

Disorders of lipid metabolism can lead to elevated levels of serum lipids, such as triglycerides and/or cholesterol. Elevated serum lipids are strongly associated with high blood pressure, cardiovascular disease, diabetes and other pathologic conditions.

Hypertriglyceridemia is an example of a lipid metabolism disorder that is characterized by high blood levels of triglycerides. It has been associated with atherosclerosis, even in the absence of high cholesterol levels cholesterolemia). When triglyceride concentrations are ive (i.e. , greater than 1000 mg/dl or 12 mmol/l), hypertriglyceridemia can also lead to atitis. Hyperlipidemia is another example of a lipid metabolism er that is characterized by elevated levels of any one or all lipids and/or lipoproteins in the blood. Current treatments for disorders of lipid metabolism, including dieting, exercise and treatment with s and other drugs, are not always ive. Accordingly, there is a need in the art for alternative treatments for subjects having disorders of lipid metabolism.

Summary of the Invention The present invention provides iRNA compositions which effect the RNA- d silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPL3 gene. The ANGPL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides s of using the iRNA compositions of the invention for inhibiting the expression of an ANGPL3 gene and/or for treating a subject who would benefit from inhibiting or reducing the expression of an ANGPL3 gene, e.g., a subject ing or prone to suffering from a er of lipid metabolism, such as a subject suffering or prone to suffering from hyperlipidemia or hypertriglyceridemia.

In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3, wherein said dsRNA comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: l and said antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5.

In another aspect, the t invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 tides from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10.

In another aspect, the present invention provides an isolated and/or non-human cell containing the dsRNA of the invention.

In another aspect, the present invention provides a vector encoding at least one strand of a dsRNA, wherein said dsRNA comprises a region of complementarity to at least a part of an mRNA encoding 3, wherein said dsRNA is 30 base pairs or less in length, and wherein said dsRNA targets said mRNA for cleavage.

In another aspect, the t invention provides an isolated and/or non-human cell comprising the vector of the invention.

In another aspect, the present invention provides a ceutical ition for inhibiting sion of an ANGPTL3 gene comprising the dsRNA of the invention or the vector of the invention.

In another aspect, the present ion es a method of inhibiting ANGPTL3 expression in an isolated and/or non-human cell, the method comprising: a) contacting the cell with the dsRNA of the invention or the vector of the invention; and b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA ript of an ANGPTL3 gene, thereby ting expression of the ANGPTL3 gene in the cell.

In another aspect, the present invention provides the use of the dsRNA of the invention or the vector of the invention in the manufacture of a medicament for the treatment of a subject having a disorder that would benefit from reduction in ANGPTL3 expression, thereby treating said subject.

Accordingly, in one aspect, the present invention provides -stranded ribonucleic acids (dsRNAs) for inhibiting expression TL3. The dsRNAs comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID N021 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5.

In another aspect, the tinvention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression ofANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10.

In one embodiment, the sense and antisense strands comprise sequences selected from the group ting ofAD-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.l, AD-53132.l, AD-52983.l, AD-52954.1, AD-52961.l, AD—52994.l, AD- 52970.l, AD-53075.1, AD-53147.l, AD-53077.1 ofTables 7 .

In certain embodiments of the invention, the dsRNAs comprise at least one modified nucleotide. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a‘ 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'—phosphorothioate group, and a terminal nucleotide linked to a teryl derivative or a dodecanoic acid ylamide group. In another embodiment, the modified tide is selected from the group consisting of a 2'- 2'-flu0ro modified nucleotide, a 2'-deoxy-modif1ed nucleotide, a locked nucleotide, an abasic nucleotide, a no-modified nucleotide, a yl-modified nucleotide, a morpholino tide, a phosphorarriidate, and a non-natural base comprising nucleotide.

The region of complementarity of the dsRNAs may be at least 17 nucleotides in length, between 19 and 21 nucleotides in length, or 19 nucleotides in length.

In one ment, each strand of a dsRNA is no more than 30 nucleotides in length.

At least one strand of a dsRNA may comprise a‘3’ overhang of at least 1 nucleotide or at least 2 nucleotides.

In certain embodiments, a dsRNA further comprises a ligand. In‘one embodiment, the ligand is conjugated to the 3’ end ofthe sense strand iof the dsRNA.

In some embodiments, the ligand is one or more ylgalactosaminc (GalNAc) derivatives attached h a bivalent or trivalent branched linker.‘ In particular'embodiments, the ligand is HO%O¢O\/\/\n/H\/l\xnlr\/?:g“ I 0 _ ,HO O\/\/\"/N/\/\N o AcHN A H H In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following schematic Viv-0.

HO;\..»1~».\.,~0-v-~,x-sp-N’, \-~’, N' *‘o ACHN 5 H H In some ments, the RNAi agent further includes at least one phosphorothioate or methylphosphonatc intemucleotide linkage. In some embodiments, the phosphorothioate or phosphonate intemucleotide linkage is at the 3’-terminal of one strand. In some embodiments, the strand is the antisense strand. In other embodiments, the strand is the sense‘strand.

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

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

In another aspect, the present invention provides a cell, e.g., a hepatocyte, containing a dsRNA of the invention.

In yet ancther aspect, the present invention es a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in , and wherein the ‘dsRNA targets the mRNA for cleavage. The region of complementarity may be least 15 nucleotides in length or 19 to 21 nucleotides in length.

In a further aspect, the present invention provides a cell sing a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA ng ANGPTL3, Wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage.

In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of an ANGPTL3 gene comprising a dsRNA or vector of the ion. lnone embodiment, the pharmaceutical composition comprises a lipid formulation, such as a MC3, SNALP or XTC formulation.

In another aspect, the present invention provideslmethods of inhibiting ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA or a vector of the ion, and ining the cell produced for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.

The cell may be within a subject, such as a human subject, for example a human subject suffering from a discrder of lipid metabolism, e.g., hyperlipidemia or hypertriglyceridemia.

In one embodiment of the methods of the invention, ANGPTL3 expression is ted 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%.

In another aspect, the present invention provides methods of treating a t having a disorder that would benefit from reduction in ANGPTL3 expression, e.g., a disorder‘of lipid lism, such as hyperlipidemia or hypertriglyceridemia. The methods e administering to the subject a therapeutically effective amount of a dsRNA or a vector of the ion, thereby treating the subject.

The disorder may be disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia In one embodiment, the administration of the dsRNA to the subject causes a decrease in the level of a serum lipid, triglycerides, cholesterol and/or free fatty acids; and/or a decrease in ANGPTL3 protein accumulation. In one embodiment, adminiStration of the dsRNA to the subject causes a decrease in the level of LDL-C, HDL-C, VLDL-C, iDL—C and/or total cholesterol.

In one embodiment, the dsRNA is administered at a dose of about 0.01 mg/kg to about 10 , e. g., about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg t 10 mgkg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mgkg to about 10.mg/kg, about 0.3 mg/kg to about 5 mgkg, about 0.3 mg/kg to about 10 mg/kg, ab0ut 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to abouth mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mgkg, 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 about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mgkg, ' about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about ‘5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mgkg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5'mgtcg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mgkg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 007,008, 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, 14,15, 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, 55, .6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this ion.

In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to ‘ about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to 1.5 about 50 mgkg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about] to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/hg, about 2.5 to about 45 mgkg, about 3 to about 45 mg/kg, about 3.5 to about 45 'mg/kg, about 4 to about 45 mgkg, 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 mgmg, 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 mgkg, about 7.5 to about 40 rug/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 mgkg, 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 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mgmg, 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 mgkg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5. to about 30 'mgkg, 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 rng/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mgmg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10' to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention. ‘ \ For example, ts can be administered a therapeutic amOunt of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1.2, 4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, ' 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12,12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the'recited values are also intended to be part of this invention.

In another aspect, the present invention provides methods of inhibiting the expression of ANGPTL3 in a subject. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby inhibiting the expression of ANGPTL3 in the subject.

In yet another aspect, the invention provides kits for ming the methods of the ion. In one aspect, the invention provides a kit for performing a method of inhibiting expression of ANGPTL3 gene in a cell by contacting a cell with a double stranded RNAi agent in an amount effective to inhibit expression of the ANGPTL3 in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to a subject.

Brief ption of the Drawings Figure l is a schematic of the experimental procedure used for in vivo tests described in Example 2. .

Figure 2, Panel A is a graph showing measured levels ofANGPTL3 n in WT mice after treatment with the indicated iRNA or a control. Figure 2, Panel B, is a graph showing measured levels of L3 proton in ob/ob mice after treatment with thc indicated iRNA or a control.

Figure 3, Panel 'A, is a graph showing ed levels of LDL-c in WT mice after treatment with the indicated iRNA or a control. Figure 3, Panel B, is a graph showing measured levels of LDL-c in ob/ob mice afier treatment with the indicated iRNA or a control.

Figure 4, Panel A, is a graph showing measured levels of triglycerides in WT mice after treatment with the indicated iRNA or a control. Figure 4, Panel B, is a graph showing ed levels of triglycerides in ob/ob mice after treatment with the indicated iRNA or a control.

Figure 5, Panel A, is a graph showing measured levels of total cholesterol (TC) in WT mice afier treatment with the indicated. iRNA or a control. Figure 5, Panel B, is a graph showing measured levels of total cholesterol (TC) in ob/ob mice after treatment with the indicated iRNA or a control.

Figure 6, Panel A, is a graph showing measured levels of HDL—c in WT mice after treatment with the indicated iRNA or a control. Figure 6, Panel B, is a graph showing measured levels of HDL-c in ob/ob mice after ent with the indicated iRNA or a control.

Figure 7 is a graph showing measured levels of ANGPTL3 protein in human PCS transgenic mice after treatment with a single dose of the indicated iRNA or a controli ed Description of the Invention The present invention provides iRNA compositions, which effect the RNA- induced silencing x (RISC)—mediated cleavage of RNA transcripts of an ANGPTL3gene. The ANGPTL3 gene may be within a cell, e.g., a cell within a subject, such as a hurnan. The present invention also provides s of using the iRNA compositions ofthe invention for inhibiting the expression of' an ANCPTL3gene and/or for treating a subject having a disorder that would benefit from ting or reducingthe expression of an ANGPTL3gene, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which, is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, '15 l5-26, 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, l8-26, 18—25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19—29, 19-28, 19-27, j 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- , 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 , which region is substantially complementary to at least part of an mRNA transcript of an ANGPTL3 gene. The use of these iRNAs enables the. ed degradation ofmRNAs of an ANGPTL3 gene in mammals. Very low dosages ofANGPTL3 iRNAs, in particular, can specifically and efficiently mediate RNA erence (RNAi), resulting in significant inhibition of expression of an 3 gene. Using cell-based assays, the present inventors have trated that iRNAs targeting ANGPTL3 can mediate RNAi, resulting in significant inhibition of expression of an ANGPTL3 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of an ANGPTL3 protein, such as a subject having a er of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia. , The following detailed description ses how to make and use compositions ning iRNAs to‘inhibit the expression of an ANGPTL3 gene, as well as compositions and methods for treating subjects having diseases and ers that would benefit from tion and/or reduction of the expression of this gene. 1. Definitions In order that the present invention may be more y understood, certain terms are first defined.- In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

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

The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to". The'term "or"- is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.- The term "ANGPTL3" refers to an angiopoietin like protein 3 having an amino acid ce from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of a native ANGPTL3.

The term encompasses full-length unprocessed precursor forms of 3 as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing of the fibrinogen-like domain. The sequence of a human ANGPTL3 mRNA transcript can be found at, for example, GenBank Accession No. G12. 41327750 (NM_ 0144952; SEQ ID NO:1). The predicted ce ofrhesus ANGPTL3 rnRNA can be, found at, for example, k Accession No. C]: 297278846 (XM_001086114.2; SEQ ID N02). The sequence of mouse ANGPTL3 rnRNA can be found at, for example, GenBank Accession No. GI: 142388354 (NM_ 0139133; SEQ ID N023). The sequence ofrat ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 68163568 (NM_001025065.1; SEQ ID N024).

The term“ANGPTL3” as used‘herein also refers to a particular polypeptide expressed in a cell by lly occurring DNA sequence variations of the ANGPTL3 gene, such as a single nucleotide polymorphism in the ANGPTL3 gene. Numerous SNPs within the ANGPTL3 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the ANGPTL3 gene may be found at, NCBI dbSNP Accession Nos. . rs193064039; rs192778191; rs192764027; rs192528948;rs191931953; rsl91293319; rsl91 171206; r3191 ; rsl91086880', rsl91'012841; or 55403.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide IS sequence of an mRNA molecule formed during the transcription of an’ANGPTLB gene, including mRNA that is a product of RNA processing of a primary transcription product.

In one embodment, the target portion of the sequence will be at least long enOUgh to serve as a ate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the ription of an 3gcne.

The target sequence may be from about 9-36 nucleotides in length, e.g., about -30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27,‘ 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15- ‘19, l5-18, l5-l7, 18-30, 18-29, 18-28, 18-27, 18-26, l8-25, 18—24, 18-23, l8-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, l9-25, 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 tides in . Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is bed by the sequence referred to using the rd tide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, e, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a d nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, ne, adenine, and uracil can be replaced by other moieties without ntially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement . For e, without limitation, a nucleotide comprising inosinc as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil,” guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide ning, for example, inosine. In another example, adenine and cytosine anywhere in the ucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target. mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,”"‘iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent thatcontains RNA as that term is defined- herein, and which mediates the targeted cleavage of an RNA transcript via an RNA- induced silencing complex (RISC) pathway. iRNA directs the sequence—specific degradation ofmRNA through a process known. as RNA interference . The iRNA modulates, e.g., inhibits, the expression ofANGPTL3 in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL3 target mRNA sequence, to direct the ge ofthc target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type II] endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-Ill-likc enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, ct al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing x (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary nse Strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15: 188). Thus, in one-aspect the invention s to a single stranded RNA (siRNA) , generated within a cell and which promotes the ion of a RISC complex to effect silencing of the target gene, i.e., an.ANGPTL3_ gene. Accordingly, the term “siRNA” is also used herein to ‘refer to an RNAi as described above.

In r aspect, the RNAi agent is a single—stranded antisense RNA molecule.

. An antisense RNA molecule is complementary to a ce within the target mRNA.

Antisense RNA can inhibit translation in a iometric manner by base pairing to the. mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355. The single-stranded antisense RNA molecule may be about 13 to about 30 nucleotides in length and have a ce that is complmentary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucledtides from one of the antisense sequences in Tables 2, 3, 7, 8, 9 and In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double-stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double-stranded RNA ) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, ed to as having ” and “antisense” orientations with respect to a target RNA, i.e., an ANGPTL3 gene. In some embodiments of the ion, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi, The duplex region may be of any length that permits c degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in , e.g., about 15-30 base pairs in length, for example, about 9, 10, ll, 12, 13, 14,15, 16, 17, 18,19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 1530, 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- , 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, 20A-24,20-23, 20- 22, 20-21, 21-30, 21-29, 21—28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths, are also contemplated to be part of the invention.

~ The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an rrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a f‘hairpin loop.” A hairpin loop can se at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise st 2, at least 3, at least 4, at least 5', at least 6, at least 7, at least 8, at least 9, at least 10, at least '20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be ntly connected.

Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective. other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA s may have the same or a different number of tides.

’ The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more tide overhangs.

As used herein, the term “nucleotide oVerhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For e, when a 3'—end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three micleotidcs, at least four nucleotides, at least five nucleotides- or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, ing a dcoxynucleotidc/nuclcoside. The overhang(s) can be on the sense strand, the nse strand or any combination thereof. Furthermore, the nucleotide(s) of an ng can be present on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1410 tide, e.g., . a l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-endland/or the 5’-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-end and/or the . In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA-mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a .25 dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. ' Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double- stranded over‘ its entire length.

The term ense strand” or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an ANGPTL3 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially mentary to a sequence, for example a target sequence, e.g., an ANGPTL3 tide sequence, as defined . Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5’- and/or 3’-terminus of the iRNA.

The term “sense strand” or nger strand" as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first tide sequencetin relation to a second nucleotide sequence, refers to the y of an oligonucleotide or polynucleotide comprising the first ‘ nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide ce, as will be understood by the skilled person. Such conditions can, for example, be stringent ions, where stringent‘conditions can 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 . Other conditions, such as logically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary ces within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” With respect to each other herein.

However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but lly not more than 5, 4, 3 or 2 ched base- pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to ize under the conditions most nt to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. r, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 tides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fiJlly mentary to the shorter oligonucleotide, can yet be referred to as “fiilly complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also e, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respectto their ability to hybridize are fulfilled. Such non—Watson-Crick base pairs e, but are not limited to, G:U Wobble or ein base pairing.

The terms “complementary,” “fiilly complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially‘ ‘ complementary to a contiguousportion of the mRNA of interest (e.g., an mRNA ng ANGPTL3). For example, a clcotide is complementary to at least a part of an ANGPTL3mRNA if the sequence is substantially complementary to a non- upted portion of an mRNA encoding ANGPTL3.

In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also e one or more non- ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA” may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in an iRNA le, are encompassed by “iRNA” for the purposes of this specification and claims.

The term “inhibiting,” as used herein, is used interchangeably with ing,” “silencing,” “downregulating,)) ‘6suppressing” and other r terms, and includes any ‘ level of inhibition.

The phrase “inhibiting expression of an ANGPTL3,” as used herein, includes tion of expression of any ANGPTL3 gene (such as, e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene) as well as variants or s of an ANGPTL3 gene that encode an ANGPTL3 protein.

“Inhibiting expression of‘an ANGPTL3 gene” includes any level of inhibition of an ANGPTL3 gene,.e.g., at least partial suppression of the expression of an ANGPTL3 gene, such as an inhibition by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at leastiabout 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%, atleast about 97%, at least about 98%, or at least about 99%.

The expression of an ANGPTL3 gene may be assessed based on the level of any variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNA level or ANGPTL3 protein level. The expression of an ANGPTL3 may also be assessed indirectly based on the levels ofa serum lipid,'a triglyceride, terol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these les compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In one embodiment, at least l ssion of the sion of an ANGPTL3 gene, is assessed by a reduction of the amount ofANGPTL3 mRNA which can be isolated from or detected in a first cell or group of cells in which an ANGPTL3 gene is transcribed and which has or have been treated such that the sion of an ANGPTL3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or_have not been so treated (control cells). The degree ofinhibition may bc’ex‘pressed in terms of: -- (mRNA in control cells) - (mRNA in treated cells) n 0100% (mRNA in control cells) The phrase cting a cell With an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. ting a cell with an RNAi agent includes contacting a cell in'vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the , or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell. ting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for e, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi . agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will uently reach the tissue where the cell to be ted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e. g., the liver. Combinations of‘in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject; . In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorptionor uptake of an iRNA can occur through unaided diffusive or active ‘ cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo uction, iRNA can be ‘ injected into a tissue site or administered systemically. In vin delivery can also be done by a beta-glucan delivery system, such as those described in US. Patent Nos. 401 and 5,607,677, and US. Publication No. 2005/0281781, the entire ts of which are hereby incorporated herein by reference. In vitro uction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

The term "SNALP" refers to a stable nucleic acid-lipid le. A SNALP is a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in US. Patent Application Publication Nos. 20060240093, 20070135372, and in '25 International Application No. WO 2009082817, the entire tsof which are hereby incorporated herein by reference. Examples of “SNALP” formulations are described below.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human e, e.g., a monkey, and a chimpanzee), a non- primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and'a whale), or a bird (e.g., a duck or a . In an embodiment, the subject is ahuman, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human having a disease, disorder or condition that would benefit from reduction in ANGPTL3 sion; and/or human being treated for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression as described herein. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result ing, such as lowering levels of triglycerides in a subject. The terms “treating” or “treatment” also include, but are not limited-to, alleviation or amelioration of one or more symptoms of a disorder of lipid metabolism, such as, e.g., a decrease in the size of eruptive xanthomas. ment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

- By “lower” in the context of a disease marker or symptom is meant a statistically - significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to'a level accepted as within the range of normal for an individual without such disorder. As used herein, ntion” or “preventing,” when used in reference to a disease, disorder or condition f, that would benefit from a reduction in expression of an ANGPTL3 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, er, or condition, (5.3., high ceride levels or eruptive xanthoma.

The likelihood of developing a'high tryglyceride levels or eruptive xanthoma is reduced, for example, when an individual having one or more risk factors for a high tryglyceride levels or eruptive xanthoma either fails to develop high tryglyceride levels or ve xanthoma or ps high tryglyceride levels or eruptive xanthoma with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction. in the development of a symptom associated with such a disease, disorder or condition i (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “serum lipid” refers to any major lipid present in the blood. Serum lipids may be present in the blood either in free form or as a part of a protein complex, e. g., a lipoprotein x. Non-limiting examples of serum lipids may include triglycerides and cholesterol, such as total cholesterol (TG), low density lipoprotein terol (LDL-C), high-density otein terol (HDL-C), very low density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C).

As used herein, a "disorder of lipid metabolism" refers to any disorder associated with or caused by a disturbance in lipid metabolism. For example, this term includes any disorder, disease or condition that can lead to hyperlipidemia, or ion characterized by abnormal ion of levels of any or all lipids and/0r lipoproteins in the blood. This term refers to an inherited disorder, such as familial hypertriglyceridemia, or an acquired disorder, such as a disorder acquired as a-result of a diet or intake of certain drugs. Exemplary disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidcmia, hypertriglyccridemia (including drug— induced hypertriglyccridemia, diuretic-induced hypertriglyccridemia, alcohol-induced ' riglyccridemia, B-adrenergic blocking induced hypertriglyccridemia, estrogen-induced hypertriglyccridemia, glucocorticoid-induced hypertriglyccridemia, rctinoid-induced hypertriglyccridemia, dine-induced hypertriglyccridemia, and al riglyccridemia), acute atitis associated with hypertriglyccridemia, chylomicron syndrom, familial chylomicronemia, Apo-E deficiency or resistance, LPL ncy or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits).

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

Disorders related to body weight are also considered “disorders of lipid metabolism”, as defined herein. Such disorders may e obesity, metabolic syndrome ing independent components of metabolic syndrome (e.g., central obesity, FBG/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, uremia, and other conditions associated with weight gain ‘ (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight regain following weight loss.

Blood sugar disorders are further considered “disorders of lipid lism”, as defined herein. Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome related to insulin resistance. Other exemplary disorders of lipid metabolism may also include renal transplantation, nephrotic syndrome, Cushing‘s syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, strophy, glycogenosis type I, and Addison's disease. peutically effective amount," as used , is intended to e the amount of an RNAi agent that, when administered. to a subject having a disorder of lipid metabolism, is sufficient to effect treatment of the e (e.g., by diminishing, rating or maintaining the existing disease or one or more symptoms of disease).

The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, , family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other dual teristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a disorder of lipid metabolism, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease ineludes slowing the course of the disease or reducing the severity of later-developing e. The "prophylactically effective amount" may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the t to be treated.

A peutically-effective " or “prophylacticaly effective amount” also es an amount, of an ,RNAi agent that produces some desired local or systemic effect at a reasonable /risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase."phannaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other m or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable carrier" as used herein 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 or zinc stearate, or steric acid),'or solvent encapsulating al, involved in ng or transporting the t compound from one organ, or n of the body, to another organ, or portion of the body. Each carrier must be table" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceu‘tically- . acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as ium state, sodium lauryl sulfate and tale; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and hylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) c acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or hydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible nces employed in ceutical formulations.

The term “sample,” as used herein, includes a tion of similar fluids, cells,- or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include s from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g.’, whole liver or certain segments of liver or n types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject.

’ II. iRNAs of the Invention Described herein are iRNAs which inhibit the expression of an ANGPTL3 gene.

In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for ting the expression of an ANGPTL3 gene in a cell, such as a be“ within a subject, e.g., a mammal, such as a human having a disorder of lipid metabolism e.g., familial hyperlipidemia. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an ANGPTL3gene, The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, or 18 nucleotides or less in ). Upon contact with a cell expressing the ANGPTL3 gene, the iRNA inhibits the expression of the ANGPTL3 gene (e.g., a human, a primate, a ' 1 non-primate, or a bird ANGPTL3 gene) by at'least about 10% as assayed by, for example; a PCR or branched DNA (bDNA)-based method, or by a protein—based method, such as by immunofluorescencc analysis, using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under ions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) es a region of complementarity that is substantially complementary, and generally fully complementary, to a target ce.

The target sequence can be derived from the sequence of an mRNA formed during the expression of an ANGPTL3gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, Such that the two strands hybridize and form a duplex structure when combined under suitable ions. As bed elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also, be ned as self-complementary regions of a single c acid. molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15—23, 15-22, 15-21, 15-20, 15-19, -18, 15-17, 18-30, 18-29, 18-28, l8-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18- , 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, 2Q-27, 20-26, 20-25, 20-24;20-23, 20-22, 20-21, 21-30, 21—29, 21-28, 21— 27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in , e.g., between 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, 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, -21, 21—30, 21-29, 21-28, 21-27, 21-26, 21-25, 21—24, 21-23, or 21-22 nucleotides in length. Ranges and lengthsintcrmediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is between about 15 and about 20 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a ate for the Dieer enzyme. For example, it is Well known in the art that dsRNAs longer than about 21—23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence- of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i. e., ge through a RJSC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about,10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14- , 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9—33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9—32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-3], 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15- 22, 15-21, 15-20, 15—19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19- 22, 19-21, _ 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. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., -30 base pairs, that s a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region r than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a lly occurring miRNA. In another embodiment, an iRNA agent useful to target ANGPTL3 expression is not generated in the target cell by ge of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1,2, 3, or 4 nucleotides. dsfiNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang'can—comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overh‘ang(s) can be on the sense strand, the antisense strand or any combination f. Furthermore the nucleotide(s) of an overhang can be present. on the 5'-end, 3'-end or both ends of either an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art-as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied tems, Inc; iRNA nds of the invention may be prepared using a two-step procedure.

First, the individual strands of the double—stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual s of the ‘\ 'siRNA compound can be prepared using solution-phase or solid-phase organic synthesis ', or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising ral or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In one , a dsRNA of the invention includes at least two tide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences ed 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 of Tables 2, 3, 7, 8, 9 and 10. In this aspect, one of the two sequences is mentary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an ANGPTL3gene. As such, in this aspect, a dsRNA will include two ucleotides, where one oligonucleotide is described as the sense strand in Tables 2, 3,7, 8, 9 and 10, and the second oligonucleotide is described 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 on separate oligonucleotides. In another embodiment, the substantially complementary ces of the dsRNA are contained on a single oligonueleotide.

' The skilled person is well aware that dsRNAs‘having a duplex structure of between about 20 and 23 base pairs, e. g., 21, base pairs (have been hailed as particularly effective in inducing RNA interference (Elbashir er al., (2001) EMBO J., 20:6877- .6888). However, others have found that r or longer RNA duplex structures can also be ive (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Na! Biotech 23:222-226). 1n the-embodiments deseribed above, by virtue of the nature of the oligonucleotide ces provided in Tables 2, 3, 7, 8, 9 and 10, dsRNAs described herein can include at least one strand of a length of lly 21 nucleotides. It can be reasonably expected, that shorter duplexes having one of the sequences of Tables 2, 3, 7, 2.0 8, 9'and 10 minusonly a few nucleotides on one or'both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 1.5, 16, l7, l8, 19, 20, or more contiguous nucleotides derived from one ofthe sequences of Tables 2, 3, 7, 8, 9 and 10, and differing in their y to inhibit the expression of an ANGPTL3gene by not more than about 5, 10, 15, 20, 25, or 30 % inhibition from a dsRNA sing the full ce, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in Tables 2, 3, 7, 8, 9 and 10 identify a site(s) in an ANGPTL3 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences ed in Tables 2, 3, 7, 8, 9 and '10 coupled to additional nucleotide sequences taken from the region contiguous to the ed sequence in an ANGPTL3gene.

While a target sequence is lly about 15-30 nucleotides in length, there is wide variation in the suitability Of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of l target sequences for any given, gene target, but an cal ch can also be taken in which a “window” or “mask” ofa given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to fy sequences in the size range that can serve as target sequences. By'moving the sequence f‘window” progressively one nucleotide upstream or downstream of an l target sequence location, the next potential target sequence can be identified, until the te set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the "identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2, 3, 7, 8, 9 and 10 representeffective target sequences, it is contemplated that r optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence fied, ag, in Tables 2, 3, 7, 8, 9 and 10, further optimization could be achieved by systematically either adding or removing nucleotides to te lenger or shorter sequences and testing those sequences generated by walking a window of the longer or r size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further ements in'the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e. g., the introduction of modified tides as described herein or as known in the art, addition or ,changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e. g., sing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An, iRNA as described herein can contain one or more mismatches to the target sequence. In One embodiment, an iRNA as described herein contains no more than 3 mismatches. lfthe antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not d in the center of the region of complementarity. .If the antisense Strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5’- or 3’-end of the region of mentarity. For example, for a 23 nucleotide iRNA agent the strand which is mentary to a region of an 3 gene, lly does not contain any mismatch within the central 13' nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an ANGPTL3 gene. Consideration of the efficacy of iRNAs » with mismatches in inhibiting expression of an ANGPTL3 gene is important, especially if the particular region of complementarity in an ANGPTL3, gene is known to have polymorphic sequence variation within the population. 111. Modified iRNAs of the Invention In one embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance‘stability or other beneficial characteristics. The c acids featured in the invention can be synthesized and/or modified by methods well ished in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby orated herein by reference. Modifications include, for example, end modifications, e.g., 5’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, DNA tides, inverted linkages, eta); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bascs that‘base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or 4’-position) or ement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments bed herein include, but are not-limited to RNAs containing modified backbones or no natural intemucleoside linkages. RNAs having modified nes include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as mes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be ucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its intemucleoside. backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl onates including 3'-alkylene phOSphonates and chiral phosphonates, phosphin‘ates, oramidates including 3'—amino phosphoramidate and lkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'—5' linkages, 2'-5'- linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Representative US. patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, US. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; ,278,302; 5,286,717; 5,321,131; 676; 5,405,939; 5,453,496; 5,455,233; ,466,677; 5,476,925; 5,519,126; 821; 5,541,316; 5,550,111; 5,563,253; ,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 639; 6,608,035; 167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 97,041,816; 7,273,933; 7,321,029; and US Pat RE39464, the entire contents of each of which are hereby orated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or eyeloalkyl intemueleoside linkages, mixed heteroatoms and alkyl or eyeloalkyl intemueleoside linkages, or one or more short chain heteroatomic or cyclie intemueleoside linkages. These'include those haying lino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; , sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkenc' containing backbones; sulfamate backbones; methylencimino and methylenehydrazino nes; sulfonate and sulfonamide backbones; amide backbones; and others having » mixed N, O, S and CH2 component parts.

Representative US. patents that teach the preparation of the above oligonueleosides include, but are not d to, US. Patent Nos. 506; 5,166,315; 5,185,444; 134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; ,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;-5,596,086; 5,602,240; ,608,046; 5,610,289; 5,618,704; 5,623,070; 312; 5,633,360; 5,677,437; and, ,677,439, the entire ts of each of which are hereby incorporated herein by- reference.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the intemucleoside linkage, i.e., the backbone, of the tide units are replaced with novel groups. The base units are maintained for ization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent ization ties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar blackbone‘of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza en atoms of the amide portion of the backbone. Representative US. patents that teach the preparation of PNA compounds include, but are not limited to, US. Patent NOS. 5,539,082; 5,714,331.; and 262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds '15 suitable for use in the iRNAs ofthe invention are described in, for example, in Nielsen er al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in ular --CH2--NH--CH2-, --CH2¥-N(CH3)--O--CH2--[known as a ene (methylimino) or MMI backbone], -—CH2--Of-N(CH3)-JCH2--, --CH2--N(CH3)--N(CH3)- '-CH2-- and --N(_CH3)--CH2--CH2--[wherein the native phosphodiester backbone is represented as -—O-—P—-O--CH2--] of the above-referenced US. Patent No. 677, and the amide backbones of the above-referenced US. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced US. Patent No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, ed herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; 0-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2),,O] mCH3, O(CH2).,,OCH3, O(CH2),,NH2, 0(CH2) nCH3, O(CH2),,ONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2' position: C. to Clo lower alkyl, substitutedvlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, , ONOz, N02, N3, Nl-lz, heterocyeloalkyl, cyeloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA ‘ cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the codynamic ties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2'—methoxyethoxy (2'-O--CH2CH20CH3,I also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'- ylaminooxyethoxy, i.e., a O(CH2)20N(CH3)2 group, also known'as 2'-DMAOE,‘ as described in examples herein belbw, and ethylaminoethoxyethoxy (also known in the art as 2'gO-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O-—CH2-- N(CH2)2.

Other modifications e 2'-methoxy (2'-OCH3), 2'—aminopropoxy (2'- OCHzCH2CH2NH2) and 2‘-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, ularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' on of 5' terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative US. patents that teach the preparation of such modified sugar structures include, but are not limited to, US. .Pat. Nos. 4,981,957; ,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 786; 785; ,519,134; 5,567,811; 5,576,427; 5,591,722; 909; 5,610,300; 5,627,053; and 5,700,920; certain ofwhich are . 5,639,873; 5,646,265; 5,658,873; 5,670,633; commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used , “unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases e (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), oxymethyl cytosine, 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-halouraeil and cytosine, ynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, il (pseudouracil), 4- acil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other S- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7—deazaguanine and 7-daazaadenine and 3-deazaguanine and 3- deazaadenine. Further nucleobases include those disclosed in US. Pat. No. 3,687,808, ' those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise opedia Of 'Polymer Science And Engineering, pages 858-859, witz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International n, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu,. B., Ed., CRC Press, 1993. Certain of these nucleobases are ularly useful for'increasing the binding affinity ofthe oligomeric nds featured in the invention. These include -substituted pyrimidines, yrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, ynyluracil and 5-propynylcytoSine. 5- methylcytosine substitutions have been shown 'to increase nucleic acid duplex stability by 0.6—1.2 °C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. entative US. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted atent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; ,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 908; 5,502,177; ,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; ,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 368; 640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to e one or more locked , nucleic acids (LNA). A locked nucleic acid is .a nucleotide having a modified ribosc moiety in which the ribosc moiety comprises an extra bridge conncCting the 2' and 4' carbons. This structure effectively "locks" the ribosc in the 3'-end'o structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J ; et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR, et al., (2007) M01 Canc Ther 6(3):833-843; Grunweller, A. el al., (2003) Nucleic Acids Research 31(12):3185-3193).

Representative US. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: US. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA les can include N— (acetylaminocaproyl)>hydroxyprolinol 6-NHAC), N-(caproyl hydroxyprolinol (Hyp-C6), N-(acetylhydroxyprolinol HAc), thymidine-2' deoxythymidine (ether), N-(aminocaproyl)-44hydroxyprolinol (Hyp-C6—amino), 2- " docosanoyl-ufidine-3"- phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. W0 20] 1/005861. 1V. iRNAs Conjugated to s Another modification ofthe RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include . but are not limited to lipid moieties such as a cholesterol moiety (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), a her, e.g., S-tritylthiol (Manoharan et al., (l992) Arm. N. Y. Acad. Sci, 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem.

LeL, 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533- '10 538), an aliphatic chain, e. g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 11-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., adecyl-rac- glycerol or triethyl-ammonium -O-hexadecyl-rac-glycerophosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651—3654; Shea et al., (1990) Nucl.

Acids Res, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) sides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan er al., (1995) edron LeII., 363651-3654), a yl moiety (Mishra el al., (1995) Biachim. Biophys. Acta,1264:229—237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp.

Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., le, cell or Cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex g in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, , an, inulin, cyclodextrin, N-acetylglucosamine, N- acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or tic molecule, such as a'synthetic r, e. g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-malcic acid anhydride copolymcr, poly(L-lactide-co-glycolicd) copolymcr, divinyl ether-maleic anhydride copolymcr, N-(2-hydroxypropyl)mcthacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N- isopropylacrylamide polymers, or polyphosphazine. e amines include: polyethylenimine, polylysine-(PLL), spermine, spermidine, polyamine, pseudopeptidepolyamine , peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic rin, nary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e. g., a lectin, glycoprotein, lipid or n, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, yl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, alent galactose, - transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, alating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic arbons (e. g., phenazine, dihydrophenazine), ial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol,-cholic acid, adamantarie acetic acid, l-pyrene butyric acid, dihydrotestosterone, 1,3-Bis- O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, l.3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3—(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabcled markers, enzymes, haptcns (e. g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e. g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole ates, Eu3+ complexes aazamacrocycles), dinitrophcnyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include es and hormone receptors. They can also include ptidic species, such as lipids, lcctins, carbohydrates, vitamins, cofactors, multivalent'lactosc, multivalent galactose, N-acetyl- galactosamine, yl—gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

' The ligand can be a substance, e. g., a drug, which can increase the uptake of the iRNA agent into the cell, for e, by disrupting the cell’s cytoskeleton, e.g., by disrupting the cell’s microtubules, microfilaments, and/or intermediate filaments. The drug canibe, for example, taxon, vincristinc, vinblastine, cytochalasin, nocodazole, inolide, ~latrunCulin A, phalloidin, swinholide A, indanocine, or myoscrvin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, es, proteinbinding , PEG, vitamins etc. Exemplary PK- modulators include, but are not d to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotidcs; e.g.,_oligonuclcotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamcrs that bind serum components (e. g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide ibed below).

This reactive oligonucleotide may be reacted directly with commercially—available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and ely made through the well-known technique of solidi-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, d Biosystems (Foster City, Calif.) Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates arid alkylated derivatives.

In the ligand-conjugated ucleotides and ligand-molecule bearing sequence- specific linked nucleosides of the present invention, the ucleotides and ucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or side conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate sors that already bear the ligand molecule, or non-nucleoside -bearing building blocks.

When using nucleotide-conjugate precursors that already bear a g moiety, the synthesis ofthe sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the g moiety to form the ligand-conjugated ucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the '. . present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid gates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule.

Such a lipid or based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a' non-kidney target tissue of the body. For example, the target tissue can be the liver, ing parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or based ligand can (a) increase resistance to degradation of the conjugate, (b) increase ing or transport into a target cell or cell membrane, and/or (0) can be used to adjust binding to a serum protein, e.g., HSA.‘ A lipid based ligand can be used to t, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney . However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other es that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for ng disorders characterized by unwanted cell proliferatiOn, e_.g., of the malignant or lignant type, e.g., cancer cells. Exemplary ns include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or lother vitamins or nutrients taken up by target cells such as liver cells. Also included are BSA and low‘density lipoprotein (LDL).

B. Cell Permeation Agents In another aspect, the ligand is a cell-permeation agent, preferably a helical cell- permeation agent. Preferably, the agent is amphipathic. An ary agent is a peptide such as tat or antennopedia. If the agent is a peptide,.it can be modified, ing a peptidylmimetic, invertomers, ptide'or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an eptidomirnetic) is a molecule capable of folding into a defined three-dimensional structure similar to anatural peptide. The attachment of peptide and , peptidomimetics to iRNA agents can affect pharrnacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, , 35, 40, 45, or 50 amino acids long.

.A peptide or peptidomimetic can be, for example, a cell pemieation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the e moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS can also be a targeting . The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and n across cell membranes. For example, sequences from the HIV Tat n (GRKKRRQRRRPPQ (SEQ ID NO: 1 1) and the Drosophila Antennapedia protein (RQlKlWFQNRRMKWKK (SEQ ID NO: 12) have been found to be capable of functioning as delivery peptides. A ' peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage=display library, or one—bead—one-compound (OBOC) combinatorial library (Larn et al., Nature, 354:82-84, 1991). Examples of a e or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic.

. A peptide moiety can range in length from about 5 amino acids'to about 40 amino acids.

The peptide moieties can have a structural modification, such as to increase stability or direct conformational ties. Any of the structural ations described below can be ed.

An RGD peptide for use in the compositions'and methods of the invention may be linear or cyclic, and may be modified, e.g., glyciosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In on to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-l or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a a;helical linear peptide (e.g., LL—37 or in Pl), a‘disulfide ontaining peptide (e.g., a -defensin, B-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation e can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide-such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen ni et al., Nucl. Acids Res. 31:2717-2724, 2003). _C. Carbohydrate Conjugates In some embodiments of the compositions and methods of the invention, an iRNA ucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as bed herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per 36 made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, en or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide'units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative ydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, en, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e. g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalacitosamine, such as H{5 an 0 AcHN 03 ‘ E HOHcfl/OMJHWNVOfl’”AcHN - HO OWu’V‘u o AcHN . 0 Formula II.

In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the ion is selected from. the group consisting of: ‘ HOHgflownwn_ AcHN OH T: ‘HOHCLéJIiOVVYHWnW/VO OAcHN O ' O O » HO - V HO OWNME o ACHN 0 Formula II, HO HO HO -0 > O\/.\O/\/0\/\N HO HO H HO '0 HO - O O\/\O/\/O\/\N,,(\/O\3JW H HO' H O O HO '0 '. O\/\O/\/O\/\N O - H Formula III, , H0 O\/\o/\/O NHAc OH \\\ HO NM HO \/0 NHAc Formula IV, NHAc , . Formula V, NHAc 1' 0 Formula Vl, HO 0M0 HO OH NHAc HO OM-O NHACHO O ”(DouayOH NHAc Formula VII, AcHN ' H a IX, AcHN H Formula X, o\/\O/\/o\/\N 0 H Formula XI, Formula XII, a X111, 0 Formula XV, OH 0 0 H fig 0 WNH OWNW H > 0 a XX, O ’ OH 0 ‘ H fig‘ 0 WNH OWNW ‘ 0 Formula XX], OH 0 .0 HOI—Eglg’HO _ O WNH ' 0 FormulaXXII.

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to; ACHN Owe/\lown_fi°H ‘ OH HO 0 HO O\/\O/\/O\/\N_n/\,O\3\NJK/\O/\/O\/\ ‘ AcHN H H . o o xo OH ~ H 4 ‘1.

HO - o , O ' QVH H0 O .

ACHN O\/\u (Forrnula XXIII), when one ofX or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate fiirther comprises one or more additional ligands as described above, such as, but not d to, a PK modulator and/or a cell permeation peptide.

D. Linkers In some embodiments, the conjugate or ligand bed herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.

The term r" or “linking group” (means an c moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, 80;, SOZNH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, kynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, rylalkenyl, rylalkynyl, larylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, a1kenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, lheterocyclylalkyl, lheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one -or more methylenes can be interrupted or terminated by O, S, 8(0), 802, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4—24, 5—24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7—17, or 8—16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is d to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e. g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference ion (which can, e.g., be selected to mimic or represent ions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage , e.g., pH, redox IO potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood.

Examples of such degradative agents include: rcdox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or ive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable g group by acting as a general acid, peptidases (which can be substrate specific), and atases.

A ble linkage group, such as a disulfide bond can be susceptible to pH.

The pH‘of human serum is 7.4, while the average intracellular pH is slightly lower, g from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at 'a preferred pl-l, thereby ing a cationic ._ lipid from the ligand inside the cell, or into the desired compartment ofthe cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleaVable linking group orated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked. to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more ntly in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable g group for the ability to resist cleavage in the blood or when in t with other rget tissue.

Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of ge in a target cell 'and the second is selected to be indicative of cleavage in other s or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In red embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular ions) as compared to blood or serum (or under in vitro ions selected to mimic extracellular conditions). i. Redox cleavable linking groups In one embodment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (78-S-). To determine if a candidate cleavable linkinggroup is a suitable “reductively cleavable linking group,” or for example is le for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the ' angwhich mimic the rate of cleavage which would be ed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In One, candidate compounds are cleaved by at most about % in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic ellular conditions) as cempared to blood (or under in vitro ions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using rd enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media. ii. Phosphate-based cleavable linking groupsln another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based- ble linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An e of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -0— Rk)—O-, -O-P(S)(ORk)-O-; )(SRk)-O;, -S—P(O)(ORk):O-, -O-P(O)(ORk)- S-, -S-P(O)(ORk)-S-, -O—P(S)(0Rk)¥S-, —S—P(S)(ORk)—O-, -O-P(O)(Rk)-O—, 40- P(S)(Rk)-O—, .—S—P(O)(Rk)-O-, —S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)( Rk)-S-.' Preferred ments are -O-P(O)(OH)—O-, -O-P(S)(OH)—O-, -O-P(S)(SH)—O-, -S- P(O)(OH)—O—, )(OH)-S-, -S-P(0)(OH)-S—, —0—P(S)(OH)—S-, -S-P(S)(OH)—O-, -O- P(O)(H)—O-, -O-P(S)(H)-O-, -S—P(O)(H)—O-, -S-P(S)(H)-O-, )(H)—S—, -O-P(S)(H)— S-. A preferred embodiment is -O—P(O)(OH)-O-. These candidates can be evaluated using methods analogous to those described above. iii. Acid cleavable linking groups . In another embodiment, a cleavable linker comprises an acid cleavable linking group; .An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In~ red embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or-lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH lles, such as cndosomes and lysosomes can provide a cleaving environment for acid cleavable linking . Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or . A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above. iv. Ester-based linking groupsln another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester- based cleavable linking groups include but are not limited to esters of ne, lene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O-, or -OC(O)-. These candidates can be evaluated using methods analogous to those bed above. v. Peptide-based cleaving grbups In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based ble linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield ‘oligopeptides (e.g., dipeptides, tripeptides etc.) and ptides. Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelenel A peptide bond is a special type of amide bond formed between amino'acids to yield peptides and proteins. The peptide based cleavage group is lly limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula — C(O)NHCHRBC(O)—, where RA and RB are the R groups of the two nt amino acids. These candidates can be evaluated using methods analogous to those described above.

In one embodiment, an iRNA'of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with'linkers of the compositions and methods of the invention include, but are not limited to, AcHN H o (Formula XXIV), HO OH 0 H AcHN H X'0 V O:0SI)=0 i IOfor 0 AcHN (Formula XXVI), HOflOMNWNYO0 H AcHN H o 2.) HO OH ”Mo—Y &\\,O\A/U\ H N O O H H N N O N —- M NO8 HO OH X=0-30 0 O H O JL y=1-15 HO OMNW - AcHN H (Formula , o 0 H .

HO OMN/VWNYO ‘ X-O AcHN H ' . 2..) ’Il/OY HO OH HO&A/ONI\NW\,N_O H N H H O N 3—Sw W0 AcHN H 16 z 0 V o x H0 0H6 x=0-30 0 H o = - 5 AcHN H (Formula XXVIII), HO OH o H v HO OMNWVNTO . X'O AcHN . H o h H& I "”O’Y H N o O OM H H H0 N - N /\,S—— w W08 AcHN ”W E wox/‘lxo . 20 y HO§O O X=1-30 H i y=1-15 H MNWN ' o z=1-20 AcHN H (Formula XXIX), and Fifi ' MON 0 O HO 5 ACHN MMWNYO NjO1/\’(O XONS Z O W0 HokowNAA/‘NAH0 0H0 x= 1-30 H o O if ::;g‘ AcHN H la XXX), when one ofX or Y is an oligonucleotide, the other is a hydrogen.

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

‘ In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of ures shown in any of formula (XXXI) — (XXXIV): a XXX] Formula XXXII P2A_Q2A_R2A}_TZA-LZA T3A_L3A ‘1 )P3A'Q3A'R3A I 3A ‘ q .

PZB-QZB‘R2B]__TZB_LZB \{\P33-Q3B-R33 ]q3—BT3F_LSB 2B PSA-Q5A_R5A T5A_L5A A-R4AErr:A-L4A qSA q PSB-QSB_R5cfl35BT58-LSB P -R L4—BT4’ 4B-Q4B 4B B_L48 PSC-QSC-RSC T5C_L5C Formula XXXIII Formula XXXIV wherein: q2A, q2B, q3A, q3B, q4A, q4B, qSA, q5B and qSC represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; PZA PZB‘ PBA P3B P4A P48 PSA PSB PSC T2A T28 T3A T313 T4A ,T4B T4A TSB TSC are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CHzNH Ol‘ CHzo; QM, QZB, QM, QBB, QM, Q43, QM, Q58, QSC are independently for each occurrence absent, alkylene; tuted alkylene wherin one or more methylenes can be interrupted or terminated by one or more of O, S, 8(0), 802, N(RN), C(R’)=C(R”), CEC or C(O); R”, R”, R”, R33, R“, R43, R“, R53, R5C are each independently for each ence absent, NH 0, s, CH2, C(O)O, , ")C(O), -C(O)—CH(Ra)-NH-, co, Miaw“r>< W W8 SW 8—8 ,W Wor heterocyclyl; LZA, LZB, L“, L”, L“, L“, LSA, LSB and L-5C represent the ligand; i.e. each independently for each occurrence a monosaCcharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andRa is H or amino acid side chain.Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such asthose of formula (XXXV): Formula xxxv -P5A_Q5A_R5A l T5A_L5A PSB-QSB-R513 1—quTSB_L53 P5C_Q5C_R5C]?T5C_L5C wherein LSA, L5“ and LSC represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II_VI], XI, X, and X11].

Representative US. patents that teach the preparation of RNA conjugates include, but are not limited to, US. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; ,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; ,109,124; 5,1 18,802; 045; 077; 5,486,603; 5,512,439; 718; ,608,046; 4,587,044; 4,605,735; 4,667,025; 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; ,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; ,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 142; 5,585,481; ,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; 8,106,022, the entire contents of each of which are hereby‘ orated herein by reference.

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

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct s, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased ance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.

An additional region of the iRNA can serve as a substrate for enzymes e of cleaving RNAzDNA or RNAIRNA hybrids. By way of e, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNAzDNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene sion. Consequently, comparable results can ofien be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to orothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the ' RNA target can be routinely detected by gel electrophoresis and, if necessary, ated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non- ligand es have included lipid moieties, such as terol (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., . Med. Chem. Lett., 1994, 4: 1053), a her, e.g., hexyl-S-tritylthiol aran et al., Ann. NY. Acad.

Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let, 1993, 322765), a} olesterol (Oberhauser et al., Nucl. Acids Res, 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras el al., EMBO J., 1991, 10:1 1 1; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk el al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or ylammonium 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), a polyamine or a hylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 142969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino—carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States s that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence.

. The amino group is then reacted with the molecule being conjugated using riate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. ation of the RNA conjugate 'by HPLC typically affords the pure conjugate. 1V. Delivery of an iRNA of the Invention The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a" disorder of lipid metabolism) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed ly by administering a composition comprising an iRNA, e.g., .a dsRNA, to a subject.

Alternatively, in viva delivery may be performed indirectly by. administering one or more vectors that encode and direct the expression of the iRNA. These atives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 39-144 and WO94/02595, which are orated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered ‘20 molecule, tion of ecific s, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local stration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the ‘expOSure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. l studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, cular delivery of a VEGF dsRNA by intravitreal injection in lgus s (Tolentino, MJ. et al., (2004) Retina 24: 132-138) and subretinal injections in mice (Reich, SJ. et al. (2003) M01. Vis. 9:210—216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume , J. et a1. (2005) Mol. Ther. 111267-274) and can prolong survival of tumor-bearing miCe (Kim, W]. et al., (2006) Mol. Ther. 141343-350; Li, S. et al., (2007) M01. Ther. 152515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dom, G. et al., (2004) Nucleic Acids 32ie49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; ra, H. et at (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA. 101217270—17275; Akaneya,Y., er al. (2005) J.

Neurophysiol. 93:594—602) and to the lungs by intranasal administration (Howard, KA. et al., (2006) M01. Ther. 14:476-484; Zhang, X. at al., (2004) J. Biol. Chem. 279:10677- 10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering an iRNA systemically‘for the treatment of a e, the RNA can be modified or alternatively delivered using a drug deliveryvsystern; both methods act to prevent the rapid . degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical ation to lipophilic groups such as cholesterol to enhance - cellular uptake and prevent degradation. For example, an iRNA ed against ApoB conjugatedto a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 3-178). Conjngation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. hnol. 24:]005—1015). In an alternative embodiment, the. iRNA can be red using drug delivery systems such as a nanopartiele, a dendrimer, a polymer, liposomes, or a cationic delivery system.

Positively charged cationic ry systems facilitate binding of an iRNA‘moleeule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA'by the cell. Cationic lipids, mers, or polymers can either be bound to an iRNA, or d to form a vesicle or micellc (see e. g., Kim SH. et al., (2008) l of Controlled Release 129(2): 107-] 16) that ericases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered ically. Methods for making and administering cationic— iRNA complexes are well within the abilities of one skilled in the art (sec e.g., Sorcnsen, DR., et at. (2003) J. Mol. Biol 1-766; 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 es of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN. et.al., (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS.‘ et al., (2006) Nature 441:111-114), cardiolipin (Chien, PY. et,al., (2005) Cancer Gene Ther. - 328; Pal, A. et al., (2005) Int]. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Plumn. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Bionzed.

Biotechnol. 7.1659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472- 487), and polyamidoamines (Tomalia, DA. et al., (2007) Biochemfloc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16721799-1804). In some embodiments, an iRNA forms a complexwith cyclodextrin for ic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in US. Patent No. 7, 427,605, which is herein incorporated-by reference in its entirety.

A. Vector encoded iRNAs of the Invention iRNA targeting the ANGPTL3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., T10. (1996), 12:5-10; Skillem, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, US. Pat. No. 299). sion can be ent (on the order of hours to weeks) or sustained (weeks to inonths or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a ar d, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid ann, er al., (1995) Proc. Natl. Acad. Sci. USA 92: 1292). ' The individual strand or strands of an iRNA can be transcribed from a er ' on an expression vector. Where two separate strands are to be sed to generate, for example, a dsRNA, two separate expression vectors can be eo—introduced (e. g., by transfection or infection) into a target cell. Alternatively each individual strand of a '10 dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynuclcotides joined by a linker polynuclcotide ce such that the dsRNA has a stem and loop structure. iRNA expression vectors are generally DNA ds or viral vectors.

Expression vectors compatible with eukaryotic cells, preferably those compatible with rate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell sion vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided ning convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA sing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the t, or by any other means that allows for introduction into a desired target cell. iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid eam'ers (e.g., Oligofeetamine) or non-eationiclipid-based carriers (e.g., Transit—TKOTM). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP).

Stable transfection of cells ex vivo can be ensured using markers that e the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin‘ B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not d to lentiviral vectors, moloney murine leukemia virus, ' 'erc.; (c) adeno- ated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) pieomavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or s adenovirus. Replication- defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells’ genome. The constructs can include viral sequences for transfection, ifdesired. Alternatively, the construct can be incorporated into vectors capable of al replication, e.g. EPV and EBV s. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc, to ensure the expression of the iRNA in target cells. Other s toconsider for vectors and constructs are further described below. s useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is ive to certain logical regulators, e.g., circulating glucose levels, or es (Docherty et al., 1994, FASEB J. 8:20-24).

Such inducible expression systems, suitable for the control of dsRNA sion in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-Dl - thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgenc.

Viral vectors that contain nucleic acid sequences encoding an iRNA can be used.

For example, a retroviral vector can be used (see Miller et al., (1993) Meth. Enzymol, 217:581-599). These retroviral vectors contain the components necessary for the cOrrect packaging of the viral genome and integration into the host cell DNA. The nucleic acid ces encoding an iRNA are cloned into one or more vectors, which facilitate delivery ofthe c acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other nces illustrating the use roviral vectors in gene therapy are: Clowcs et al., (1994) J. Clin. Invest. 93:644-651; Kicm et al., (1994) Biood 83: 1467-1473; s and rg,’(l993) Human Gene Therapy 4: 129-141; and Grossman and Wilson, (1993) Curr. Opin. in Genetics and Devel. 3:110-114. Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in US. Patent Nos. 6,143,520; 5,665,557; and ,981,276, which are herein incorporated by reference.

Adenoviruscs are also contemplated for use in delivery of iRNAs of the invention. Adenoviruscs are especially attractive vehicles, e. g., for delivering genes to respiratory epithelia. Adenoviruscs lly infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, elial cells, and muscle. Adenoviruses have the advantage of being e of infecting non-dividing cells. Kozarsky and Wilson, (1993) Current Opinion in Genetics and Development 503 present a review of adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy 5:3-10 trated the use of adenovirus vectors to transfer genes to the respiratory lia of rhesus‘monkeys.

Other instances 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 682143-155; Mastrangeli et al., (1993) J. Clin. Invest. 91:225-234; PCT Publication 2649; and Wang et a1., (1995) Gene Therapy 22775-783. A suitable AV vector for expressing an iRNA ed in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. E: 1006-1010. associated virus (AAV) vectors may also be used to ry an iRNA of the invention (Walsh e1 al.,-(1993) Proc. Soc.'Exp. Biol. Med. 9-300; US. Pat.

No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV , and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J; Virol. 61: 3096-310]; Fisher [(1 et al. (1996), J. Virol, 70: 520-532; Samulski R et a1. (1989), J. Virol. 63: 3822-3826; US. Pat. No. 5,252,479; US. Pat. No. 5,139,941; tional Patent Application No. WO 94/13788; and International Patent Application No. WC 93/2464], the entire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inevtion is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens'from other viruses, or by substituting different viral capsid proteins, as appropriate. For e, lentiviral vectors can be pseudotyped with surface proteins from vesicular itis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the s to express different capsid protein serotypes; see, e.'g., Rabinowitz J E et a1.

, J Viral 76:791-801, the entire disclosure of which is herein incorporated by reference.

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

V. Pharmaceutical Compositions of the Invention ' The - present invention also includes pharmaceutical compositions and formulations which e the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharrnaceutically acceptable carrieri The pharmaceutical itions containing the iRNA are useful for treating a disease or disorder associated with the expression or. activity of an ANGPTL3 gene, e.g., a disorder of lipid metabolism, such as hypertriglyceridemia.

Such pharmaceutical compositions are formulated based on the mode of delivery.

One example is'compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) or for subcutaneous delivery. Another- example is itions that are formulated for direct delivery into the liver, e.g., by infiision into the liver, such as by continuous pump infusion.

The pharmaceutical itions of the invention may be administered in dosages sufficient to inhibit expression of a ANGPTL3 gene. In general, a le dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of ~25 .about'l to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mgkg, about 0.05 mg/kg, about 0.5 mykg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mgkg, about 20 mg/kg, about mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8.1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4,‘5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

In r embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to'about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to abOut 50 mg/kg, about 15 to about 50 mgkg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to {about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mgkg, about 4 to about 45 mg/kg, about 4.5 to about 45 mgkg, 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 mgkg, 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 mgkg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mgkg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mgkg, about 0.5 to about 40 mgkg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/rng, 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 abouti40 mgkg, about 7.5 to about 40,mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 05 to about 30 mg/kg, about 0.75 . to about 30 mg/kg, about 1 to about 30 mg/mg, about l.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 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mgkg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 201mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mgkg, about 7.5 to about 20 mg/kg, about 10 to about 20 mgkg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, .4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7', 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3,‘8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this ion.

In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mgkb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mgkg, about 4.5 to '30 about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about ‘50 mg/kg, about 15 to about 50 mgkg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mgkg, about 1 to about 45 mg/mg, about l.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 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 mgkg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about ‘45 mgkg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 rug/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, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 ‘mg/kg, about 20 to about 40 mykg, 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 0.5 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 mgkg, 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 mgkg, 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 mgkg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to. about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mgkg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mgkg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values-and ranges intermediate to the recited values are also ed to be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9,1, 1.1,1.2, l.3,1.4,1.5,1.6,1.7,18. 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, 38 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, 14,145,15,15.5,l6,16.5,17,17.5,l8,18.5,19,19.5,20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more ses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each se must be correspondingly , smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period.

Sustained release formulations are well known in the art and are particularly useful for- delivery of agents at a particular site, such as could be used with the agents of the t invention. In this embodiment, the dosage unit ns a corresponding multiple of the daily dose.

The effect of a single dose on ANGPTL3 levels can be long lasting, such that uent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors can influence the dosage . and timing ed to effectively treat a subject, including but not d to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, ent of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in viva testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as. disorders of lipid metabolism that would ' benefit from reduction in the sion of ANGPTL3. Such models can be used for in vivo testing of iRNA, as well as for determining a eutically ive dose.

Suitable mouse models are known in the art and include, for example, an obese (ob/ob) mouse containing a mutation in the obese (ob) gene ( Wiegman et al., (2003) Diabetes, 52: 1081-1089); a mouse containing homozygous knock-out of an ‘LDL receptor (LDLR -/- mouse; shi et al., (1993) J Clin Invest 883-893); diet-induced artherosclcrosis mouse model (Ishida et al., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoprotein lipase knockout mouse model (Weistock et al., (1995) J . Clin.

Invest. 96(6):2555-2568).

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by ncbulizer; intratracheal, intranasal, epidermal and ermal, oral or parenteral: Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathccal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular , such as the liver (e.g., the hcpatocytes' of the liver).

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, s and s. Conventional pharmaceutical carriers, aqueous, powder or x oily bases, thickeners and the like can be necessary or desirable. Coated cOndoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical ry agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., diolcoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl ol DMPG) and cationic (e.g., yltetramethylaminopropyl DOTAP and diolcoylphosphatidyl ethanolamine DO'l‘MA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to ic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to ic lipids. le fatty acids and esters include but are not limited to arachidonic acid, oleic acid, noic acid, lauric acid, caprylic acid, capric acid, myristic acid, ic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl l-monocaprate, l- dodecylazacycloheptan-Z-one, an acylcamitine, an acylcholine, or a C1-2o alkyl ester (e.g., isopropylmyrlstate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical forrnulations‘are described in detail in US. Patent No. 6,747,014, which is orated herein by nce.

A. iRNA Formulations Comprising Membranous Molecular Assemblies An iRNA for use- in the compositions and methods of the invention can be formulated for delivery in a membranous lar assembly, e.g., a liposome or a -micelle. As used herein, the term “liposome” refers to.a vesicle composed of hilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. mes include ellar and multilamellar vesicles that have a membrane formed fiom a lipophilic material and an aqueous interior. The aqueoUs portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which lly does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ients to the site of action. Because the liposomal membrane - is structurally similar to ical membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses 'with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous ts that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by avariety of methods. In one example, the lipid component of a liposome is ved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an athic cationic lipid or lipid ate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAiagent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.

If necessary a carrier nd that assists in condensation can be added during . the condensation reaction, e.g., by lled addition._ For example, the r compound can be a polymer other than a nucleic acid (e.g., sperrnine or spermidine). pH can also adjusted to favor sation.

Methods for producing stable‘polynucleotide delivery vehicles, which . incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are fiirther described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in r, P. L. et al., (1987) Proc. Natl.

Acad. Sci. USA 8:7413-7417; US. Pat. No. 4,897,355; US. Pat. No. 5,171,678; Bangham. et al., (1965) M Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys.

Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 7. Commonly used techniques for preparing lipid ates of riate size for use as delivery 'vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim.

Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew at al., (1984) Biochim.‘ , Biophys. Actq 775: 169. These methods are readily d to ing RNAi agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the vely d nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell - cytoplasm (Wang el al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

Liposomes, Which are pH-sensitive or negatively d, entrap nucleic acids rather than complex with them. Since both the’nucleic acid and the lipid are similarly charged, repulsion rather than complex formation . heless, some nucleic acid is ped within the aqueous interior of these mes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou el al. (1992) Journal ofControlled Release, 19:269-274).

One‘ major type of liposomal composition es phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or itoyl atidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is fornicd from phosphatidylcholine.(PC) such as, for example, soybean PC, and egg PC. Another type is formed from es of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other s to introduce liposomes into cells in uitro and in vivo include US. Pat. No. 5,283,185; US. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; W0 91/16024; Felgner, (1994) J. Biol. Chem. 26912550; Nab‘el, (1993) Proc. Natl.

Acad. Sci. 90111307; Nabel, (1992) Human Gene Ther. 3:649; n, (1993) Biochem. 3227143; and Strauss, (1992) EMBO J. 11:417.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems sing non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylenestearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylenestearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal s were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P.Pharma.'Sci., 4(6):466).

Liposomes also include cally stabilized” liposomes, a term which, as used herein, refers to mes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to mes lacking such specialized . Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as aloganglioside G M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) - moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing iosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized ' liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES). (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 5323765); Various liposomes comprising one or more glycolipids are known in the art.

Papahadjopoulos et al. (Ann. N. Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside GM], galactocerebroside sulfate and atidylinositol to improve blood half-lives of liposomes. These findings were ded upon by '20 Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85,:6949). US. Pat. No. 028 and WO 88/04924, both to 'Allen et al., disclose liposomes comprising (1) sphingomyclin and (2) the ganglioside GM. or a galactocerebroside sulfate ester. US.

Pat. No. 5,543,152 (Webb er al.) discloses mes comprising sphingomyelin.

Liposomes comprising 1,2.—sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage g able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as ntly with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes ed from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from lism and degradation (R’osoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker , 1988, volume 1, p. 245). Important erations in the preparation of liposome formulations are the lipid surface charge, e size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[l -(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid—nucleic acid xes which are capable of fusing with the negatively d lipids .of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and US. Pat. No. 4,897,355 for a description ofDOTMA and its use with DNA).

A DOTMA analogue, l,2-bis(oleoyloxy)(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. LipofectinTM Bethesda Research tories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise vely charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive.

Positively charged complexes prepared in this way neously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, l,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, a) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other ed cationic lipid compounds include those that have been conjugated . to a variety of moieties including, for example, carboxyspermine which has been conjugated to one oftwo types of lipids and includes compounds such as 45— carboxyspermylglycine dioctaoleoylamide (“DOGS”) (TransfectamTM, Promega, Madison, Wisconsin) and dipa1mitoylphosphatidylethanolamine 5-carboxyspermyl- amide (“DPPES”) (see, e.g., US. Pat. No. 5,171,678).

Another ic lipid conjugate es derivatization ofthe lipid with - cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280).

Lipopolylysine, made by conjugating polylysine to DOPE, has been reportedto be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim.

Biophys. Acta 1065z8). For certain. ce1l lines, these liposomes containing conjugated cationic lipids, are said to exhibit-lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products e DMRlE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA)I(Life Technology, Inc, Gaithersburg, Maryland). Other cationic lipids Suitable for the delivery of ucleotides are bed in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for l administration, liposomes present several advantages over other ations. Such ages include d side s related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) l ofDrug Targeting, vol. 410 and du Plessis et al., (1992) Antiviral ch, 18:259-265; Mannino, R. J. and Fould- ' Fogerite, S., (1998) hniques 6:682—690; Itani, T. ez al., (1987) Gene 562267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine their utility ~ in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-lO-stearyl ether) and Novasome II (glyceryl distcaratc/ cholcstcrol/polyoxycthylene-lO-stcaryl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder. mes that include iRNA can be made'highly able. Such , deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of able liposomes. Transferosomes can be made by adding surface. edge activators, y surfactants, to a standard liposomal ition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to ' deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transderrnal gradient. In addition, due to the lipid properties, these transferosomes can be ptimizing (adaptive to the shape of pores,,e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other ations amenable to the present invention are described in United States provisional application "serial Nos. 61/018,616, filed January 2, 2008; 61/018,61 l, filed January 2, 2008; 61/039,748, filed March 26, 2008; 61/047,087, filed April 22, ’ 2008 and 61/051,528, filed May 8, 2008. PCT application no , filed October 3, 2007 also describes formulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Trans‘fersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets t fragmenting, and often self-loading. To make transfersomes it is le to add surface edge-activators, usually surfactants, to a standard liposomal composition.

Transfersomes have been used to deliver serum n to the skin. The transfersome— mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find‘wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations r, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.

Nonionic tants find wide application in pharmaceutical and cosmetic products and are usable over a wide range ofpH . In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl , polyglyceryl esters, sorbitan esters, sucrose , and lated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylatcd alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The yethylene surfactants are the most popular members of the nonionic surfactant class.

If the tant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants e carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl es, sulfonates~ such as alkyl benzene ates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.

The most ant members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammoniumsalts and ethoXylated amines. The quaternary ammonium salts are the most used members of this class. , lfthe surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as eric. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkylbetaines and phosphatides.

The use-of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in PharmaceuticalDosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285.). - The iRNA for use in. the methods of the invention can also be provided as ar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in‘a spherical structure Such that all the hydrophobic portions of the molecules are directed inward, leaving the hilic ns in contact with the surrounding aqueous phase. The converse ement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal nes may be prepared by mixing an s solution of the siRNA ition, an alkali metal C3 to C22 alkyl sulphate, and a micelle forming nds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically able salts of hyaluronic acid, ic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.

The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to e smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl te. The first micellar composition is then mixed with at least three micelle g compounds to form a mixed micellar - composition. ln another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining e forming compounds, with us mixing.

Phenol and/or m-cresol may be added to the mixed ar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as in may also be added after formation of the mixed micellar composition. -25 For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior toydispensing a n of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray, Propellants may include hydrogen-containing chlorofluoroearbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (l ,l ,l ,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ients can be ined by relatively straightforward experimentation. For absorption through the oral cavities, it is ofien desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Nucleic acid lipidparticles iRNAs, e.g., dsRNAs of in the inVention may be fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. - As used herein, the term "SNALP" refers to a stable nucleic acid-lipid le,-including '15 SPLP. As used , the term "SPLP" refers to a nucleic acid-lipid. particle comprising plasmid DNA ulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid ate). SNALPs and SPLPs are extremely useful for systemic applications, as they t extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the stration site). SPLPs include ," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No.

~ WO 00/03683. The particles of the present invention typically'have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 1 10 nm, most typically about 70 nm to about 90 nm, and. are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid- lipid particles of the present ion are resistant in s solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., US. Patent Nos. 5,976,567; 501; 6,534,484; 410; 6,815,432; US. Publication No. 2010/0324120 and PCT ation No. WO 96/40964.

In one ment, the lipid to drug ratio (mass/mass ratio) (e. g., lipid to dsRNA ratio) _will be in the range of from about 1:1 to about 50:1, from about 1:] to ' about 25:1, from about 3:1 to about 15:], from about 4:1 to about 10:1, from about 5:] to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl—NN-dimethylammonium. chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l - (2,3- dioleoyloxy)propy1)—N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)-N,N,N-trirnethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxY)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N—dimethylaminopropane (DLinDMA), linolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2- Dilinoleylcarbamoyloxydimethylaminopropane (DLin-C—DAP), 1,2-Dilinoley0xy—3- (dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3—morpholinopropane (DLin-MA), l,2-Dilinoleoyldimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio- 3-dimethylaminopropane S-DMA), 1-Linoleoyllinoleyloxy dimethylaminopropane (DLin-Z-DMAP), 1,2-Dilinoleyloxytrimethy1aminopropane chloride salt (DLin-TMA.C1), l,2-Di1inolcoyltrimcthy1aminopropane chloride salt (DLin-TAP.C1), 1,2-Dilinoleyloxy(N-methylpiperazino)propane (DLin-MPZ), or 3- (N,N-Dilinoleylamino)-1 ,2-propanediol (DLinAP), -Dioleylamino)-1,2- propancdio (DOAP), 1,2-Dilinoleyloxo(2—N,N-dimcthylamino)ethoxypropane (DLin-EG-DMA), 1,2—Dilinolenyloxy—N,N-dimethylaminopropane (DLinDMA), 2,2- Dilinoleyldimethylaminomethyl-[ l ,3]—dioxolane (DLin-K-DMA) or analogs thereof, (3aR,55,6aS)—N,N-dimethyl-2,2-di((9Z,1 ZZ)-octadeca-9,12-dienyl)tetrahydro-3a1 cyclopenta[d][1,3]dioxolamine (ALN 100), (6Z,9Z,28Z,31Z)—heptatriaconta- ,31-tetraenyl 4-(dimethylamino)butan0ate (MC3), 1,1'—(2-(4-(2-((2—(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin- l — yl)ethylazanediyl)didodecanol (Tech G1), or. a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle; In another embodiment, the compound 2,2-Dilinoleyldimethylaminoethyl- [1 ,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2- Dilinoleyldimethylaminoethyl-[1,3]—dioxolane is described in ' States provisional patent application number ,998 filed on October 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl dimethylaminoethyl-[l ,3]—dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C- DOMG (molepercent) with a particle size of 63.0 :t 20 nm and a 0.027_siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine , ylphosphatidylcholine , dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol , dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-pho'sphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POP-E), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE- mal), dipalmitoyl phosphatidyl lamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16 dimethyl PE, 18-] -trans PE, 1 -stearoyl-2:oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about. 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. .25 The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)—1ipid including, without limitation, a acylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG- dilauryloxypropyl (Ciz), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Cig), or a PEG- ryloxypropyl (C]g). The conjugated lipid that prevents aggregation of les can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %_of the total lipid present in the particle.

In one embodiment, the lipidoid ND98-4HCI (MW I487) (see US. Patent Application No. ,230, filed 008, which is orated herein by reference), terol (Sigma-Aldrich), and PEG-Ceramide Cl 6 (Avanti Polar Lipids) can be used to prepare lipid—dsRNA nanOpartieles (i.e., LNPOl particles). Stock ons of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG— Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded h a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion Step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, is or tangential flow filtration. Buffer can be exchanged with,’for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. 0 NW H i H ' /\/\/\/\/\/\ Jl\/\ /\/N\/\ /\/N N\/\/\/W\/ 11 N N: “fir H H ND98 Isomer l Formula 1 LNPOl formulations are described, e.g_., in lntemational Application Publication No. , which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA ations are described in the table ' below. ic lipid/non-cationic ' lipid/cholesteroVPEG-lipid Ionizable/Cati onic Lipid conjugate ‘ Lipid:siRNA ratio DLinDMA/DPPC/Cholesterol/PEG- CDMA l,2-Dilinolenyloxy-N,N- . dimethylaminopropane (DLinDMA) 7.1/34.4/1 .4) ‘Iipid:siRNA ~ 7:1 XTC/DPPC/Cholesterol/PEG- ‘ 2,2-Dilinoleyldimethylaminoethyl—[l,3]— cD_MA dioxolane (XTC) 57.1/7.1/34.4/l.4 lipid:siRNA ~ 7:1 XTC/DSPC/Cholesterol/PEG—DMG 2,2-Dilino|eyldimethylaminoethyl-[1,3]- 57.5/7.5/3l.5/3.5 ane (XTC) lipid:siRNA ~ 6:1 “ XTC/DSPC/Cholesterol/PEG-DMG 2,2-DilinoleyI-4—dimethylaminoethyl-[1,3]- dioxolanc (XTC) 57.5/75/3 l .5/3.5 lipid:siRNA ~ 11:1 XTC/DSPC/Cholcsterol/PEG-DMG 2,2-Dilinoleyldimethylaminoethyl-[1,3]- 60/7.5/3]/] .5, dioxolane (XTC) lipidzsiRNA ~V6:1 XTC/DSPC/Cholesterol/PEG-DMG 2,2-Dilinoleyldimethylaminoethyl—[1,3]— 60/7.5/31/1.5, . dioxo1ane (XTC) lipidzsiRNA ~ 11:] XTC/DVSPC/Cholcsterol/PEG—DMG 2,2-Dilinoleyldimethylaminoethyl—[1,3]- dioxolane (XTC) 50/10/385/15 Lipid:siRNA 10:1 ALN] OO/DSPC/Cholesterol/PEG— (3aR,SS,6aS)-N,N-dimethyl-2,2-di((9Z,122)- .DMG octadeca~9,12-dienyl)tetrahydro-3aH- 50/10/38.5/l .5 cyclopcnta[d][1,3]di0xol-5'—aminc (ALNl 00) Lipid:siRNA 10:1 SPC/Cholesterol/PEG- (6Z,9Z,282,31Z)-heptatriaconta—6,9,28,31 - DMG- n- 1 9-yl ethylamino)butanoate 50/10/38.5/1.5 (MC3) Lipid:siRNA 10:] Tech Gl/DSPC/Cholcsterol/PEG- 1, ] '-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2- hydroxydodecy|)amino)ethyl)piperazin 50/10/38.5/1.5 yl)cthy]azancdiyl)didodccanol (chh GI) Lipid:siRNA 1031 PC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 'MC3/DSPC/Chol/PEG— DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 MC3/DSPC/Chol/PEG-DSG LNP17 MC3 50/10/38.5/1.5 I ' . MC3/DSPC/Chol/PEG-DMG LNP18 MC3 50/10/385/115 _ ‘ LipidzsiRNA: 12:] MC3/DSPC/Chol/PEG-DMG LNP19 MC3 50/10/35/5 LipidzsiRNA: 8:] MC3/DSPC/Chol/PEG-DPG, LNP20' MC3 . 50/10/38.5/1.5‘ . O/DSPC/Chol/PEG-DSG LNP21 c12—200 50/lO/38..5/l.5 XTC/DSPC/Chol/PEG-DSG LNP22 50/10/38.5/1.5 siRNA: 10:] DSPC: distcaroylphosphatidylchol inc DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (Cl4-PEG, or PEG-C14) (PEG with avg molwt of 2000) PEG-DSG: styryl glycerol (C18—PEG, or PEG-C18) (PEG wi_th avg mol wt of 2000) PEG-CDMA: PEG-carbamoyl-l ,2-dimyristyloxypropylaminc (PEG with avg mol wt of 2000) SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) compri'sing ations are described in International Publication No. W02009/ 127060, filed April , 2009, which is hereby incorporated by reference.

‘XTC comprising formulations are described, e.g., in US. Provisional Serial Nol 61/148,366, filed January 29, 2009; US. Provisional Serial No. 61/156,851, filed March 2, 2009; US. Provisional Serial No. filed June 10, 2009; US. Provisional Serial No. 61/228,373,'filed July 24, 2009; US. Provisional Serial No. ,686, filed September 3, 2009, and lntemational Application No. ,fi1ed January 29,2010, which are hereby orated by reference.

MC3 comprising formulations are described, e.g., in US. Publication No. 2010/0324120, filed June 10, 2010, the entire contents of which are hereby incorporated by reference.

ALNY-lOO comprising ations are described, e.g., International patent . application number PCT/USO9/63933, filed on November 10, 2009, which is hereby incorporated by reference.

C12-200 comprising formulations are described in US. Provisional Serial No. 61/ 0, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of ionizable/cationic lipids " Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid- lipid particles of the inventiOn can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise. ” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated ht chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls e cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacenticarbon atoms. Alkenyls e both cis and trans isomers.

Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1- butenyl, 2-butenyl, isobutylenyl, l-pentenyl, 2—pentenyl, 3-methyl-l -butenyl, 2-methyl- . 2-butenyl, 2,3-dimethy1butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally ns at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls e acetylenyl, propynyl, l-butynyl, 2-butynyl, ynyl, 2- yl, 3-methyl-l butynyl, and the like.

“Acyl” any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, - ' C(=O)alkyl, -C(=O)alkenyl, and -C(=O.)alkynyl are acyl .

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to_ lO-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or ic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and Sliiflll' heteroatoms can be optionally ed, and the nitrogen heteroatom can be optionally quatemized, including ic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be ed via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidihyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted cycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo tuent (=0) two hydrogen atoms are replaced. In this'regard, substituents include oxo, halogen, heterocycle, -CN, -ORx, -NRny, -NRxC(=O)Ry, -NRxSOZRy, -C(=O)Rx, ORx, -C(=O)NR_ny, — SQnRx and -SOnNRny, wherein n is 0, 1 or 2, Rx and Ry are the same or ent and independently hydrogen, alkyl or heterocyele,land each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, -OI-l, -CN,i alkyl, ~0Rx, heterocyclc, -NRny, -NRxC(=O)Ry, -NRxSOZRy, -C(=O)Rx, -C(=O)ORx, -C(=O)NRny, jSOnRx and -SOnNRny.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use of protecting groups. Protecting group ology is well known to those d in the art (see, for example, Protective Groups in Organic sis, Green, T.W. er al., Wiley- lnterscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting grOup” is used. An “alcohol ting group” is any group which decreases or- eliminates unwanted reactivity of an l functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of a A In some embodiments, nucleic acid-lipid particles of the invention are formulated ’ using a cationic lipid of formula A: where R] and R2 are independently alkyl, l or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyldimethylaminoethyl-[l,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Scheme 1 Br OH ‘ Br . _ )‘k , 1 3 4 2 O R NHR R —-————> 2 -—» ' R R‘ R2 1 O 4 3 Formula A Lipid A, where R1 and R2 are independently alkyl, l or l, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of l and 2 yields ketal 3. Treatment of ketal 3 withamine 4 yields lipids of formula A. The lipids of formulaA can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, e, or the like.

Scheme2 Bng—R1 + Rz-CN L O=<R2 Alternatively, the ketone 1 starting material can be prepared according to Scheme 2.. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. '10 Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1 .

.S'ynthesis ofMC3 Preparation of DLin-M—C3-DMA (i.e., (6Z,9Z,282,3lZ)-heptatriaconta- 6,9,28,31-tetraen-l9-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,2SZ,3lZ)—heptatriaconta-6,9,28,31-tetraenol (0.53 g), 4-N,N- dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and l-ethyl-3—(3-dimethylaminopropyl)carbodiimide hloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid ed by dilute aqueous sodium bicarbonate.

The organic fractions were dried over ous magnesium sulphate, filtered and the t removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. FraCtions ning the purified product were combined and the solvent removed, yielding a colorless oil (0.54 Synthesis ofALNY-100 Synthesis of ketai 519 [ALNY-lOO] was performed using the ing scheme ”“300 NHMe NCsze .sNCbZMe NCsze ”MO- 0504 ‘ LAH Cbz-OSu, NEI3 + _’ ——"’ —_'_“' H0 ” 5‘6 515 511A 511BOH o l PISA MezNuu<Io — ’ . — LAH.1MTHF o — <——_ o _ — MECDZNIU'CE.o — . 513 Synthesis 0f515 To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (IL), was added a solution of 514 (10g, 0.04926mol) in 70 mL ofTHF slowly at 0°C under nitrogen atmosphere. Afier complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the e ' was cooled to 0 0C and quenched with l addition of saturated NaZSO4 solution.

Reaction mixture was stirred for 4 h at room ature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL e and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to fumish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g lH-NMR (DMSO, 400MHz): 5: 9.34 (broad, 2H), 5.68 (5’. 2H), 3.74 (m, IH), 2.66-2.60 (m, 2H), 2.50-2.45 (m, SH).

Synthesis of 51 6 To a stirred on of compound 515 in 100 mL dry DCM in a 250 mL two neek RBF, was added NEI} (37.2 mL, 0.2669 mol) and cooled to 0 OC under‘nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)—succinimide (20 g, 7 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) e was washed successively with 1N HCl solution (1 x 100 mL) and saturated NaHC03 solution (1 x 50 mL). The organic layer was then dried over anhyd. Na2$04 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). lH-NMR (CDClg, 400MHz): 5 = 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%).

Synthesis of 5] 7A and 51 7B The Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N—oxide (7.6 g, 0.06492 mol) ed by 4.2 mL of 7.6% on of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (~ 3' h), the mixture was quenched with addition of solid Na2S03 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2 x 100 mL) followed by saturated NaHC03 (1 x 50 mL) solution, water (1 x 30 mL) and finally with brine (1x 50 mL). Organic phase was dried over an.Na2804 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: - 6 g crude 517A - Peak-l (white solid), 5.13 g (96%). lH-NMR (DMSO, 400MHz): 6: 7.39—7.3l(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, 7.86%. chemistry confirmed by X-ray.

Synthesis of 51 8 Using a procedure ous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR(CDC13, ): 5= 7.35-7.33(m, 4H), .27(m, 1H), 5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m,] H), 4.58-4.57(m,2H), 2.78-2.74(m,7H), 2.06-2.00(m,8H), 1.96-1 .91(m, 2H), l.62(m, 4H), , 2H), 1.37-1 .25(br m, 36H), , 6H). HPLC-98.65%.

General Procedurefor the Synthesis of Compound 519 A solution of compound 518 (1 eq) in hexane (1'5 mL) was added in a drop—wise fashion to an ice-cold solution ofLAH in THF (1 M, 2 eq). Afier complete addition, the .10 e was heated at 40 °C over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR 8 = 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): Molecular weight for C44H30N02 (M + H)+ Calc. 654.6, Found 654.6. ations prepared by either the rd or extrusion-free method can be characterized in similar s. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvem Zetasizer Nano ZS (Malvem, USA). Particles should be about 20-300 nm, such as 40-100 mm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, ‘ as well asthe entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 mm,- at least 90 nm, at least 100 nm, at least 110 nm, and at least-120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or es, microparticulates, nanoparticulates, suspensions or solutions in water or non- aqueous media, capsules, gel. capsules, sachets, tablets or minitablets. Thickeners, flavoring , diluents, emulsifiers, sing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the _ invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable tants include fatty acids and/or esters or salts f, bile acids and/or salts thereof. Suitable bile acids/salts e chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro—24,25- dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitie acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, ein, dilaurin, glyceryl l-monocaprate, 1-dodecyla2acycloheptan-Z- one, an acylcamitine, an acylcholine,or a monoglyceride, a diglycen'de or a pharmaceutically acceptable salt thereof (e. g., sodium). In some ments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA: Further penetration enhancers include ‘ yethylenelauryl ether, polyoxyethylene-ZO-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form ing sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly- amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyallcylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylatcs; DEAE-derivatized polyimincs, pollulans, celluloses and starches. Suitable complexing agents e chitosan, N-trimethylchitosan, poly-LLIysinc, polyhistidine, polyomithine, polysperrnincs, protaminc, polyvinylpyn'dine, polythiodiethylaminomethylethylenc P(TDAE), inostyrene (e.g., p-amino), p0ly(methylcyanoacrylate), poly(cthylcyanoacrylatc), poly(butylcyanoacrylate), poly(isobutylcyanoacrylatc), DEAE- _ poly(isohcxylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, acrylamidc, DEAE-albumin and. extran, polymethylacrylate, xylacrylate, poly(D,L-lactic acid), poly(DL—lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in US. Patent 6,887,906, US Publn. N0. 27780, and US Patent No. 6,747,014, each of.which is incorporated herein by reference.

Compositions and formulations for parenteral, arenchymal (into the brain), intrathecal, intraventricular or epatic administration can e sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, r compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-eontaining formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. ularly preferred are formulations that target the liver when treating hepatic disorders such as , hepatic carcinoma.

The pharmaceutical ations of the present invention, which'can ' conveniently be presented in unit dosage form, can be prepared ing to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of ng into association the active ingredients with the ceutical carrier(s) or excipient(s). In general, the formulations are ed by uniformly and tely bringing into association the active ingredients with liquid rs or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not d to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in s, non-aqueous or mixed media. .10 Aqueo‘us suspensions can further contain substances which increase the viscosityvof the suspension including, for example, sodium carboxymethyleellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional ations i. .Emulsions The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1um in diameter (see e. g., Ansel's , Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., eh 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, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., in Remington‘s Pharmaceutical es, Mack Publishing Co., Easton, Pa., 1985, p. 30]). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. ln general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is . finely d into and dispersed as minute droplets into a'bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can - be present as a solution in either aqueous phase, oily phase or itself as a separate phase.

Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are sed of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o)- and water-in-oil-in-water (w/o/w) emulsions, Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil ts of an o/w emulsion enclose small water droplets conStitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o. emulsion.

Emulsions are characterized by little or no thermodynamic stability. Ofien, the dispersed or discontinuous phase of the emulsion is well sed into the external or uous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the ease of emulsion-style ointment bases and creams. Other means of izing emulsions entail the use of fiers that can be orated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e. g., s Pharmaceutical Dosage Forms and Drug ry Systems, Allen, LV., ch 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).

Synthetic surfactants, also known as surface active agents, have found wide ability in the formulation of emulsions and have been ed in the ture (see e.g., s Pharmaceutical Dosage Forms and Drug ry Systems, Allen, LV., ch NG., and Ansel HC., 2004, cott Williams & s (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker . (Eds), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), Marcel , Inc., New York, N.Y., 1988, volume I, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrOphobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic. group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker ~ 0 (Eds), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include n, x, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as ite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and idants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; ldson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, eenan, guar gun, karaya gum, and tragacanth), cellulose tives (for example, carboxymethyleellulose and carboxypropylcellulose), and synthetic rs (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to ‘form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the ity of the external phase.

Since emulsions ofien contain a number of ingredients such as carbohydrates, proteins, s and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used vatives included in ~ emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, konium chloride, esters of p-hydroxybenzoic acid, and boric acid.

Antioxidants are also ly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free l scavengers such as toeopherols, alkyl gallates, butylatcd hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and idant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippineott Williams & Wilkins (8th ed), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral ry have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., 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, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, man, Rieger and Banker (Eds), 1988, Marcel Dekker, Inc, New York, N.Y., volume 1, p. 199). Mineral-oil'base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions. ,ii. Microemulsions In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can, be defined as a , system of water, oil and amphiphile which is a single optically pic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery s, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in ceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-lengthalcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropicaily clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 5). mulsions commonly are ed via a ation of three to five components that include oil, water, surfactant, cosurfaetant and electrolyte. Whether the microemulsion is of the water—in-oil (w/o) or an oil—in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical es, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to - formulate microemulsions (see e.g., 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, N.Y., volume 1, p. 245; Block, in ceutical Dosage Forms, Lieberman, Rieger and Banker (Eds), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p'. 335).

Compared to conventional emulsions, microemulsions offer the age of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. tants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML3lO),.tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol scquioleate ($0750), decaglycerol eatc (DA0750), alone'or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, l-propanol, and nol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be ed t the use of cosurfactants and alcohol-free mulsifying microemulsion systems are known in the art. The s phase can typically be, but is not limited to, water, an aqueous solution of the drug, ol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300,>Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolizcd C8-C lO glycerides, ble oils and ne oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., US. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.

Find. Exp. Clin. col, 1993, 13, 205). Microemulsions afford advantages of ,10 improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations _in membrane fluidity and bility, ease of ation, ease of oral administration over solid dosage forms, ed clinical potency, and decreased toxicity (see e. g., US. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides at al., Pharmaceutical- Research, 1994, 11, 1385; Ho etal.,.1. Pharm. Sci, 1996, 85, 138-143). Often ' microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, es or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and ceutical applications. It is expected that the miCroemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and c acids.

Microemulsions of the t invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers rove the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad eategories--surfactants, fatty acids, bile salts, ing , and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above. iii. Microparticles an RNAi agent of the ion may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other s including lyophilization, evaporation, fluid bed drying, vacuum drying, or a‘combination of these techniques. iv. Penetration Enhancers In one embodiment, the present ion employs various ation ers ' to effect the efficient delivery Of nucleic acids, ularly iRNAs, to the skin of animals. Most drugs are present in on in both ionized and nonionized forms.

However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. in addition to aiding the ‘ diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad ries, i.e., surfactants, fatty acids, bile salts, chelating agents, and non—chelating non-surfactants (see e. g., en, M. Surfactants and polymers in drug delivery, '20 lnforma Health Care, New York, NY, 2002; Lee et al., Critical Reviews in eutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail. tants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous on and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, yethylenelauryl ether and polyoxyethylene-ZO-cetyl ether) (see e.g., Malmsten, M. Surfactants and rs in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in eutic Drug Carrier Systems, 1991, p.92); and rochemical ons, such as FC-43. Takahashi er al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which 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, ate, tricaprate, monoolein (l-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1- monocaprate, 1—dodecylazacycloheptan—2-one, acyleamitines, acylcholines, €1-20 alkyl esters thereof (e. g.-, methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, e, atc, palmitate, stearatc, linolcate, etc.) (see e.g., Touitou, E., el al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006', Lee er al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; E1 Hariri et al., J.

Pharm. Pharmacol, 1992, 44, 651—654).

The physiological role ofbile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed, Hardman el al. Eds, McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration ers. Thus the term "bile salts" includes any of the naturally ing components of bile as well as any of their synthetic tives. Suitable bile salts include, for example, cholic acid (or its pharrnaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium idehydrocholate), holic acid (sodium holate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxyeholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylenelauryl ether (POE) (see e. g., Malmsten, M. tants and polymers in drug delivery, Informa Health Care, New York, NY, 2002', Lee et (11., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: ton's Pharmaceutical Sciences, 18th Ed., Gennaro, cd., Mack Publishing Co., Easton, Pa., 1990, pages 3; 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).

Chelating agents, as used in connection with the present invention, can be IO defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced.

With regards to their use as penetration enhancers in the present invention, Chelating agents have the added advantage of also serving‘as DNase inhibitors, as most characterized DNA nucleases e a divalent metal ion for catalysis and are thus inhibited by Chelating agents tt, J. Chromatogr., 1993, 618, 9). 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 tives of collagen, h—9 and N—amino acyl tives of beta—diketones (enamines)(see e.g., Katdare, A. et al., ent developmentfor pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee at 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).

As used herein, elating rfactant ation enhancing compounds can be defined as compounds that demonstrate insignificant activity as Chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1—33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, l-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et " 116 . al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991 , page 92); and non- dal anti-inflammatory agents such as diclofenac , indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, US. Pat. No. 5,705,188), cationic ol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Examples of cially ble transfection reagents include, for example LipofectamineTM (lnvitrogen; Carlsbad, CA), Lipofectamine 2000TM (Invitrogen; Carlsbad, CA), 293fcctinTM (Invitrogcn; Carlsbad, CA), CcllfectinTM (Invitrogen; Carlsbad, CA), DMRlE—CTM (Invitrogen; ad, CA), Freestyle“? MAX rogcn; Carlsbad, CA), LipofectamineTM 2000 CD (Invitrogcn;‘Carlsbad, CA), LipOfectamineTM (Ilnvitrogcn; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oli'gofcctamincTM '15 (lnvitrogen; Carlsbad, CA), Optifect'l‘M (lnvitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP mal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (GrenZacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFastTM Transfection Reagent (Promega; Madison, WI), foTM-ZO Reagent (Promega; Madison, WI), foTM-SO t (Promega; Madison, WI), DreamFectTM (OZ Biosciences; Marseille, ), EcoTransfect (OZ Biosciences; Marseille, France), TransPass" D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVecTM/LipoGenTM (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER ection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent ntis; San Diego,.CA, USA), BaculoPORTER ection Reagent ntis; San Diego, CA, USA), TroganPORTERTM ection Reagent (Genlantis; San 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 TM (B-Bridge International, Mountain View, CA, USA), among others.

Other agents can be utilized to enhance the penetration of the administered c acids, including glycols such as ethylene glycol and propylene , pyrrols such as 2—pyrrol, azones, and terpenes such as ne and menthone. v. Carriers Certain compositions of the present invention also incorporate carrier nds in the formulation. As used herein, “carrier nd” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological ty per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration ofa nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.

For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran e,_polycytidic acid or 4-acetamido-4'isothiocyano-stilberie-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. J vi. Excipients In st to a carrier compound, a “pharmaceutical carrier” or “excipient” is a aceutically acceptable solvent, suspending agent or any other cologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of stration in mind, so as to e for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. l pharmaceutical carriers include, but arenot limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, eta); fillers (e.g;, lactose and other sugars, microerystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylatcs or calcium hydrogen phosphate, eta); lubricants (e.g.., magnesium stearate,. talc, silica, colloidal silicon dioxide, stearie acid, ic stearates, hydrogenated vegetable oils, corn , polyethylene glycols, sodium benzoate, sodium e, etc.); disintegrants (e. g., starch, sodium starch glycolate, etc.); and wetting agents (e. g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic cxcipients suitable for non- eral administration which do not deleteriously react with c acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically » acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicie acid, viscous n, hydroxymethylcellulose, polyvinylpyrrolidone and the like; Formulations for tdpical administration of nucleic acids can include sterile and erile aqueous solutions, non-aqueous solutions in common solvents such as, alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic cxcipients suitable for non-parenteral administration which do- not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable exeipients include, but are not d to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, visccus n, ymethylcellulose, polyvinylpyrrolidone and the like. vii. Other Components The compositions of the present ion can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art- ' established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example,»antipruritics,l astringents, ‘local anesthetics or anti—inflammatory agents, or can n additional materials useful in physically formulating various dosage forms of the compOSitions of the present ion, such as dyes, ng agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of theipresent invention. The formulations can be sterilized and, if desired, mixed with'auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing c pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the '15 nucleic ) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain izers.

In some embodiments, pharmaceutical itions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi ism and which are useful in ng a disorder of lipid metabolism.‘ Examples of such agents include, but are not lmited to an anti-inflammatory agent, anti- steatosis agent, iral, and/or anti-fibrosis agent. In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include udine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., US. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and‘in Hale et al., US. Application Publication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures-in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the tion). The dose ratio n toxic and therapeutic effects is the therapeutic index and it can be sed as the ratio LDso/EDso. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell e assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the EDso With little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically ive dose can be estimated initially from cell culture assays. A dose can be ated in animal models to achieVe a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the 'IC50 (i.e., the concentration of the test compound which achieves a half-maximal tion of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in ation with other known agents effective in treatment of pathological ses mediated by ANGPTL3 expression. In any event, the stering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. Methods of the Invention The present invention also provides s of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene in the cell, ion in gene expression can be assessed by any methods known in the art. For example, a reduction in the sion of ANGPTL3 may be determined by determining the mRNA expression level of ANGPTL3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of ANGPTL3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques. A reduction in the expression ofANGPTL3 may also be assessed indirectly by measuring a decrease in biological activity of 3, e.g., a decrease in the level of serum lipid, triglycerides, cholesterol and/or free fatty acids.

In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell le for treatment using the s of the invention may be any cell that expresses an ANGPTL3gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a nonehuman primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, amouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (etgi, a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

ANGPTL3 expression is inhibited in the cell by at leaSt about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%.

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least apart of an RNA transcript of the 3 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, intrapcritoneal, or parenteral routes, including ranial (e. g., , intraventricular, intraparenchymal and intrathecal), intravenous, uscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and l (including buceal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, ‘ e.g.., a desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. A depot injection may also e more consistent serum concentrations. Depot injections may include aneous injections or uscular injections. In preferred ments, the depot ion is a subcutaneous injection.

In seme embodiments, the administration is via a pump. The pump may be an external pump or aisurgically ted pump. In certain embodiments, the pump is a subcutaneously implanted, osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically ted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In'one aspect, the present invention also provides methods for inhibiting the expression of an ANGPTL3 gene in a mammal. The methods include stering to the mammal a ition comprising a dsRNA that targets an ANGPTL3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript or the ANGPTL3 gene, thereby inhibiting sion of the ANGPTLIi»gene in the-cell. Reduction in gene expression can be ed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e. g. ELISA, bed herein. In one embodiment, a puncture liver ~ biopsy sample serves as the tissue material for monitoring the reduction in ANGPTL3 gene and/or protein expression.

The present invention r provides methods of treatment of a subject in need thereof. The treatment methods of the invention e administering an iRNA of the ion to a subject, e.g., a subject that would benefit from a reduction and/or inhibition of ANGPTL3 sion, in a therapeutically effective amount of an iRNA targeting an ANGPTL3 gene or a pharmaceutical composition comprising an iRNA targeting an ANGPTL3 gene.

An iRNA of the invention may be administered as a “free’iRNA.” A free iRNA is administered in the e of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity ofthe buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an'iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression are those having a disorder of lipid lism, e.g., an inherited disorder of lipid metabolism or an, acquired disorder of lipid metabolism. In one embodiment, a subject having disorder of lipid metabolism has hyperlipidemia. In another ment, a subject having a disorder of lipid lism has IO hypertriglyceridemia. Treatment of a subject that would t from a reduction and/or inhibition of ANGPTL3 gene expression includes therapeutic treatment (e.g., a subject is having eruptive xanthomas) and prophylactic treatment (e.g., the subject is not having eruptive xanthomas or a subject-may be at risk of developing eruptive xanthomas).

The invention filrther provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known phamaceuticals and/or known eutic methods, such as, for e, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting 3 is administered in combination with, e. g., an agent useful in treating a disorder-of lipid metabolism as described elsewhere herein. For e, additional agents suitable for treating a subject that would benefit from reducton in ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, may include agents that lower one or more serum lipids.

Non-limiting examples of such agents may e cholesterol synthesis tors, such as HMG-CoA reductase inhibitors, e.g., Statins. Statins may e atorvastatin (Lipitor), fluvastatin l), lovastatin (Mevacor), lovastatin extended-release (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crcstor), and simvastatin (Zocor). Other agents useful in treating a disorder of lipid lism may include bile sequestering agents, such as cholestyramine and other resins; VLDL ion inhibitors, such as niacin; lipophilic antioxidants, such as ol; acyl-CoA terol acyl transferase inhibitors; id X receptor antagonists; sterol regulatory binding protein cleavage activating protein (SCAP) activatOrs; omal ceride transfer protein (MTP) inhibitors; elated peptide; and therapeutic dies against ANGPTL3. The additional therapeutic agents may also include agents that raise ' high density lipoprotein (HDL), such as cholesteryl ester transfer protein (CETP) inhibitors. Furthermore, the additional therapeutic agents may also include dietary supplements, e.g., fish oil. The iRNA and additional therapeutic agents may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or deseribed herein. .

In one embodiment, the method includes administering a composition featured - herein such that expression of the target ANGPTL3 gene is decreased, such as for about 1,2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours,'28, 32, or abour 36 hours. In one embodiment, expression of the target ANGPTL3 gene is decreased for an extended duration, e. g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target ANGPTL3gene.

Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as bed herein.

Administration of the dsRNA according to the methods of the invention may result in a reduction of the ty, signs, symptoms, and/or s of such diseases or disorders in a patient with a disorder of lipid metabolism. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be ed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being . treated or ed for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by ing any one-of such parameters, or any combination of parameters. For e, cy of treatment of a disorder of lipid metabolism may be assessed, for example, by periodic monitoring of one or more serum lipid levels . Comparisons of the later readings with the initial readings provide a physician an indication of r the treatment is effective. It is well within the ability of one skilled in the an to monitor efficacy of treatment or'prcVCntion by measuring any one of such parameters, or any combination of ters; In connection with the administration of an iRNA targeting ANGPTL3 or pharmaceutical composition thereof, "effective against" a disorder of lipid metabolism indicates that administration in a ally appropriate manner results in a beneficial effect for at. least a statistically significant fraction of ts, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating er of lipid metabolisms and the related causes.

A treatment Or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to p symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and ably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of ent is ced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by areduction in the severity of e as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity g scale, as but one example the Child-Pugh score (sometimes the Child-Turcotte-Pugh score). Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.

Subjects can be stered a therapeutic amount of dsRNA, such as about 0.01 mgkg to about 5 mg/kg, about 0.01 rug/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mgkg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, 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 mgkg, about 0.5 mg/kg to about 10 mg/kg, about 1 mgkg 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 mgjkg to about 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/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 mgkg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about‘10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to‘about 10 mg/kg, or about 9.5 mgkg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be'part . of this invention.

For example, the dsRNA may be administered at a dose of about 0. 1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1,9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 61,62, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg.

. Values and ranges intermediate to the recited values are also ed to be part of this invention.

In other embodiments, for example, when a composition of the invention comprises a dsRNA as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount‘of dsRNA, such as a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mgmg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 , about 7.5 to about 50 mg/kg, about 10 to about 50 mgkg, 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 rug/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50. I mgkg, 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 mgmg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 rng/kg, about 3 to about 45 rug/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 mgkg, 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’, 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 mgkg, 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 trig/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 mgkg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about mgkg, 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 ' mgkg, 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 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For e, subjects can be administered a therapeutic amount of dsRNA, such as'about 0.1, 0.2, 0.13, 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, 24, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16.5,17,17.5,18,18.5,19,19.5,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8,9,10,11,12,13,14,15,16,17,18,19, 20,21, 22, 23, 24, or about a 25 minute period. The administration may be ed, for example, on a r basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months'or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after stration biweekly for " three , administration can be repeated once per month, for six months or a year or longer. Administration of the iRNA can reduce ANGPTL3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, , 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 at least about 99% or more, Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and red for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unWanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the iRNA can be administered aneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time, such as over 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 days. The administration may be ed, for‘ example, on a r basis, such as biweekly (i.e., every two weeks), for one month, two months, three months, four months or longer. Afler an initial treatment regimen, the treatments can be stered on a less freQuent basis. In some embodiments, a single dose of iRNA is followed by monthly dosing. In some ments, the dosing may comprise a loading phase of multiple doses on consequitive days.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ry skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be' used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by nce in their entirety. In case of conflict, the present specification, including definitions, will l. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES e ‘1. iRNA Synthesis Source of reagents Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts siRNA design was carried out to identify siRNAs targeting the human ANGPTL3 transcript annotated in the NCBI Gene database //wwwncbi.nlm.nih.gov/gene/) and a lgus monkey (Macaca fascicularis; orth “cyno”) ANGPTL3 transcript produced via sequencing of cDNA prepared from liver RNA. Sequencing of cyno ANGPTL3 mRNA was done in-house, and the mRNA sequence is shown in SEQ' ID NO:9.‘ Design used the following transcripts from the NCBI collection: .Human - NM_014495.2 (SEQ ID NO:1) ; Mouse - NM_013913.3 (SEQ ID NO:2). All siRNA duplexes were ed that shared 100% identity with the listed human and cyno ‘20’ transcripts. A subset of siRNA duplexes, described below, also shared 100% identity with the mouse (Mus musculus) ANGPTL3 transcript found in NCBI Gene database. siRNA Design, Specificity, and Ejficacy Prediction The ted specificity of all possible 19mers was predicted from each sequence. Candidate l9mers were then selected that lacked repeats longer than 7 nucleotides. These 977 candidate human/cyno , and a subset of 38 that also matched mouse (“human/cyno/mouse candidate siRNAs”) were then used in a hensive search against the human transcriptome (defined as the set ofNM_ and XM_ records within the human NCBI Refseq set) using an exhaustive ‘fbrute-force” algorithm implemented in the python script ‘BruteForcepy’. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential 'off-target’ transcript. The off-target score is ed to emphasize differences in the 'secd' region of siRNAs, in positions 2- 9 from the 5' end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12—19 counted as 1.0. An additional off- target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5’ start were used to create 2 heptamers and one octomer.

‘Heptamerl ’ was created by adding a 3’ A to the hexamer; ‘heptamer2’ was created by adding a 5’ A to the hexamer; octomer was created by adding an A to both 5’ and 3’ ends of the hexamer. The frequency ofoctomers and heptamers in the human 3’UTRome (defined as the subsequence of the riptome from NCBI’s Refseq database where the end of the coding region, the ‘CDS’, is clearly defined) was pre- calculated. The octomer fi'equency was ized to the heptamer frequency using the - median value from the range of octomer frequencies. A ‘mirseedScore’ was then ated by calculating the sum of ( (3 X normalized octomer count ) + ( 2 X heptamerZ count) + (1 X erl count)).

Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 es as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately c. Sorting was carried out by the specificity of the antisense . es were then selected from the human/cyno set with nse oligos g miRNA seed matches, scores of 3 or better, less than 65% overall GC content, no GC at the first position, 4 or more Us or As in the seed region, and GC at the nineteenth position. es from the human/cyno/mouse set with antisense oligos having scores of 2 or better, less than 65% overall GC content, and no GC at the first position were also selected. siRNA ce selection A total of 47 sense and 47 antisense derived siRNA oligos from the human/cyno set were synthesized and formed into duplexes. A total of 15 sense and 15 antisense derived siRNAs from the human/cyno/mouse set were synthesized and formed "into es.

Synthesis L3 sequences ANGPTL3 sequences were synthesized on a MerMade 192 synthesizer at either a l or 0.2 pmol scale. Single strands were synthesized with 2’O-methyl modifications for transfection based in vitro screening; For use in free uptake screening assays, 3’ GalNAc conjugates were made with 2’F and 2’-O-methyl chemical modifications. In these s, GalNAc moiety was placed at the 3’end of the sense strand. The antisense sequence was 23 nucleotides in length and also contained 2’F and 2’Omethyl chemical modifications with two phosphorothioate linkages at the 3’end.

On one set of 21mer single strands and duplexes, ‘endolight’ chemistry was applied as detailed below. 0 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) 0 In the antisense strand, pyrimidines adjacent (towards 5’ position) to ribo A nucleoside were replaced with their corresponding 2’-O-Methyl nucleosidcs 0 A two base dedT extension at the 3’ end of both sense and anti sense sequences was introduced For GalNAc conjugated 21mer sense and complementary 23mer antisense ces, 2’F and 2’0Methyl modified single strands were synthesized. The synthesis was performed on a GalNAc modified CPG support for the sense strand and CPG d with universal support for the antisense sequence at a 1 ,umol scale. The sequence motif named TOFFEE was applied, in which the sense strand contained a three-nucleotide 2’F7modified motif at positions 9, 10 and 11 and in the antisense, a 2’OMethyl-modified motif was included at positions 1 l, 12 and 13. sis, Cleavage and Deprotectjon The synthesis ofANGPTL3 sequences used solid supported oligonucleotide' synthesis using phospho‘ramidite chemistry. For 21 mer endolight sequences, a deoxy thymidine CPG Was used as the solid support while for the ’GalNAc conjugates, GalNAc, solid support for the sense strand and a universal CPG for the antisesense strand were used.

The synthesis of the above sequences was med at either a l or 0.2 pm scale in 96 well plates. The amidite ons were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrilc) was used as the activator.

The synthesized ces were cleaved and deprotected in 96 well plates, using . methylamine in the first step and fluoride reagent in the second step. For GalNAc and 2"F nucleoside ning sequences, deprotection conditions were modified. Sequences afier cleavage and deprotection were precipitated using an acetone: l (80:20) min and the pellets were re-suspended in 0.2M sodium acetate buffer. s from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a ' selected set of samples by IEX chromatography to determine purity.

Purification, Desalting and Annealing ANGPTL3 sequences were precipitated and purified on an AKTA Purifier system using a Sephadex column. The ANGPTL3 was run at ambient temperature.

Sample injection and collection was performed in 96 well plates with 1.8 mL deep wells.

A single peak corresponding to the full length sequence was collected in the eluent. The desalted ANGPTL3 sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The complementary single strands were then combined in a 1:1 sto‘ichiometric ratio to form siRNA es.

Example 2. In vitro screening Cell e and transfections Hep3B cells (ATCC, Manassas, VA) were grown to near nce at 37 °C in an atmosphere of 5% C02. in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfcction was carried out by adding 14.8 ul of Opti-MEM plus 0.2 ul of Lipofectaminc RNAiMax per well (Invitrogcn, ad CA. cat # 13778—150) to 5 ul of siRNA duplexes per well into a 96-well plate and incubated at room temperature for minutes. 80 ul of complete growth media without antibiotic containing ~2 x104 l-lep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose ments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at , l, 0.5, 01,005, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless ise stated.

Free uptake transfection '5 ul of each, GalNac conjugated siRNA in PBS was combined with 4X104 freshly thawed cryopreserved lgus monkey hepatocytes resuspended in 95 ul of In Vitro Gro CP media (In Vitro Technologies- Celsis, Baltimore, MD) in each well of a 96 well plate. The mixture was incubated for about 24 hrs at 37 °C in an atmosphere of % C02. siRNAs were tested at final concentrations of 500nM, lOOnM and 10nM for efficacy free uptake assays. For dose response screens, final siRNA concentrations were 500nM, lOOnM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM and 0.0064nM.

Total RNA isolation using DYNABEADS mRNA Isolation Kit (lnvitrogen, part #.' 6/0— Cells were harvested and lysed in 150 pl of Lysis/Binding Buffer then mixed for minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten iters of magnetic beads and 80 pl of Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the ing beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 pl Wash Buffer A and mixed for 1 . Beads were captured again and supernatant removed. Beads were then washed with 150 pl of Wash Buffer B, captured, and the supernatant was removed. Beads were next washed with 150 pl n Buffer, captured, and the supernatant was removed. Beads were allowed to dry for 2 minutes. After drying, 50 pl of Elution Buffer was added and mixed for 5 minutes at .70 °C. Beads were captured on magnet for 5 minutes. 40 pl Of supernatant was d and added to r 96 well plate. cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied tems, Foster City, CA, Cat #4368813) A master mix of2 pl 10X Buffer, 0.8 pl 25X dNTPs, 2 pl Random primers, 1 pl Reverse Transcriptase, 1 pl RNase inhibitor and 3.2 pl of H20 per reaction were added - into l0 pl total RNA. cDNA was generated using a Bio-Rad C-lOOO or 8-1 000 thermal cycler (Hercules, CA) through the following steps: 25 °C 10 min, 37 0C 120 min, 85 0C sec, 4 °C hold.

Real time PCR 2 pl of cDNA was added to a master mix containing 0.5 pl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 pl ANGPTL TaqMan probe (Applied Biosystems cat # H30020558l_ml) and 5p] Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat # 04887301001). Real ' time PCR was done in an AB] 7900HT Real Time PCR system (Applied tems) using the AACt(RQ) assay. Each duplex~ was tested in two ndent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To ate relative fold change, real time data was analyzed using the AACt method and ized to assays performed with cells transfected with 10 nM AD-l 955, or mock transfected cells. leos were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-l 955 or naive cells over the same dose range, or to its own lowest dose. 'AD-l955 sequence, used as a negative control, .15 targets luciferase and has the following sequence: sense: uGAGuAcuchAdedT (SEQ ID NO: 14); antisense: UCGAAGuACUcAGCGuAAGdedT (SEQ ID NO: 15).

Viability screens ' Cell viability was measured on days 3 'and 6 in HeLa and Hep3B cells following transfection with 10, 1, 0.5, 0.1, 0.05 nM siRNA, Cells were plated at a density of ,000 Cells per well in 96 well plates. Each siRNA was assayed in triplicate and the data ed. siRNAs targeting PLKl and AD-l 9200 were included as positive controls for loss of viability, and AD-l 955 and mock transfected cells as negative controls. PLKl and AD-l9200 result inla dose dependent loss of viability. To measure viability, 20 pl of CellTiter Blue (Promega) was added to each well of the 96 well plates afier 3 or 6 days and incubated at 37 °C for 2 hours. Plates were then read in a ‘ Spectrophotometer (Molecular Devices) at 590Em. Viability was expressed as the average value of light units from three ate transfections +/- standard deviation.

Relative viability was assessed by first averaging the three replicate transfections and then normalizing Mock transfected cells. Data is expressed as % e cells.

Table 1: Abbreviations of nucleotide monomers used in nucleic acid sequence representation.

It will be tood that these monomers, when present in an oligonucleotide, are mutually linked by 5'—3'--phosphodiester bonds Abbreviation 528a ~338|22 . mmméwm mewDV mmwfivm mmonvoH mH~-me mfivéov momémw ona-NmH wHoHéooH Noméwv Noméwv hHmAmmm oHvH-Nmma whm-mmm 8.633% 552633. 52-2 Amucfimwnnm 3535‘ yo 82 .296 o. E 33 <3<<<u2<osu<o<<<<<3 3333,}? uuooosa<<33<usoa<a< ou3<<u8<<8288< 039920858803 825330338532: u<ooa<8302<§3<<o3 38838333050: 253383: o<a=o<==o<<=a<<oaaa os<<33<<onsoa<ou3< ODDDD<U<<UD<OOD<OOD DD<D<DDDO<DGDDU<ODD 321% 85m==< 2.82 mAPLUZ< 0Nom-< Hdeomi . “—o SE8; N33812: Ea5ga;Ea5g3gEé5%55 83$ 833 :3me CENTNmmH whmémm n . $32.35. .

“Esta 8533 .33 Awucmfiwnnm 8:855». “3 35m no: :68 ES a. 3:3 83 <2338353<o33<3 <<38<<8<<8<<uw<o 3<2<u<03<<33<<5uoo 223533303350 255838533838 3825325385 535335503830 3823032033 6333335550 <<<uaz<<33u<<3<<8 <<<uaa<<38<<8<<8 33855033535 <UUD<UUD<ODDOD<<<<U <<UDO<<U<DU<<<D<D<U Ecficfisi E$5.». H.mONmm-< H.mvam-< 2an .5330 EaEa5%5%555%EaEa55EaEa55 H.mww.mv-o< fimmwmvfiz hmw-mmw OHOH-Nmm omNH-N\.NH 26-5mm mmoHSHoH Nmfiéofi Nehéwo Sofimmm mmfih: 83$ mm~-wm~ mmNéNN vmonHOH 83-83 vnmémm mwménm mONH-wwHH mNhé: mmNHfimNH U3000300330<0<0330< 00<<UUU00<3U303303< 2288330383: 8353353038: w3<3538o3<o<33< 3325883233: 0308:2352??? u<<00000<3u303303<3 0003333<033U3<<<U0< nos<3<<<ua<oau<o<<< 3w3<2<<<un<08<o<<< 2538335283: 83863803323: 9:38:33328383 3303322850322 UUU3000<<U3<003033< <<0<3 0<<UUU3< 330300300330330<30< <UU3U333303300<333< fidmaomé‘ H.0mmom-< E5EEEE fi.owflwm-< fivfimmmk‘ H.w:om-< uq -< H.O¢Nom-< hmw-mmw OHOH-Nmm omflfifl 32.22 333 83$ HHOH-mmm moHAvH 832. 839 wmgNN £8-88 83-wa Emémm. wwmémm oONH-wwHH mNné: mm~Tmn~a <<U3U3U<<00<UUU<0 <0<3000003300 <Bu<<u<<<<o<woawao 60226333356 35835353335 3835233838 83883335555 <3<0<<0<0<3UU000330 300333<0<<U3<<<<UUO 38305333232 5283053533332 <2<83038<o<<<38 <<83<<uo<<uao<35u 5852835625 ao<<u<8<<<3<=<u<8 3<<0<003<033000<000 3<000330<3U33 330300 3U<3U<<0<<UU<U0<0<< 3<<<3UU<<U<<<<0<003 H.mmamm-< fimmwomi Ea 5% HmHNom< HNDwmi H.mmfiom-< H.mN~om-< H.mm~wm-< floommvé/w Ea 5% 5%fimmaomé‘ H.mNmmv-o< Hznommvé< Hmomm¢3< EaEaEa5%5%Ea v-o< H.~ommv-o< v.o< H.mmmmv-o< wmvéwv Nomaéwmfi HwH-moH DOWNQm HONH-mw: H353” moaéma Ham-mmw 05-me Hum-mmm Hmm-mHm mmwnfifiwfi oom-~w~ momémw mhfim vabNN NmoHéHOH om~-w¢m ov©--m 83853338335. 523333 uo<3<u<<8383<o<3 000308333<<<o<o<3< un<<<8<u<<<<o<su<< oaunuoonsaz<onaa<< 332538330233 85385335383 u3<<<uo§<u<<8283 53382333320: 8338382383: o<<u<<33o<u<330< 3<<<u3<08<w<<<<<3< <oos<8203<<3<<oas o2o23<<380<8<<o<< 3<<<uz<08<o< 05038638833 uow333<<3<uaoa§<< 033038336333: Eé5%a;5gEaEEEgEg5g5g5gEaEa5Ea!EB555g3% 3<<38<<8<<8<<uo 3<3<<§ooa<<<3u<<u 8280 3<8833<<<o<u<u8 38<3u3383823<o u3<<<<uuo<w<u 30380333523 <u<<una<<3<u<oo3<u 3233838935 <3<2<u<3<<o <<<u<up<ooa<o<a<<ou 8<<<88<<<<3038 32.333830630233 <<u33<§3<u<ooz<83 3833838233296 .8863033323 8353533388 23<3<u<u2<<3<<<8o <<a<<<uno<3<um<u<<u EaEE3%EaEaEaEaEaEaEa3%3%EaEa5%EEEEH.mNmm.v-o<, Sowmvg 323?? 333-2 MNHHLmOHH mmwhom om~-~H~ HmNHLmnwfi NH mmoéwo m~m$om mmvaé 3A ASN-9; Nwaévfi 83302823833 08388038335 88383353883 63883332033033: 388238230333: 0033080383233: 83838338383 83333238833: 033302833833 8:882:38833 Ea5g5%5%5%5%5g53EgEE 5338 582833802338 33333828338 3835333830: 35835333830 <3<<833u33<8 3833358335 <3<83<<<33<<8 3383336335 333633833 %5%3%5%5%EaEa5%5%Ea $32683. mucwacwmwmcwmzca. .822 “35533 82 530 9 E Ba pvflu<=<<<3<oa<o<<<<<z HUmHUUDUODDO<JDO<330<<33 HUmHUUUOODDD<<JD<uDOD<3< HUmhuGUDaguDuguUDDUDOx‘ 0<00<<onu<<uuusoz< Dw<<uDDG<ODDO<ODDD <GODSUDGD<<JD<<OD HuthDD<GDDUD<<<uw<3<u<< puflugaaaauiésogw: +ufluw<30<30<<3<<033 $£8<So<30<<3<<w32 Fufluoa<<=3<<onaon<822< Sfl83333<u<<5<wos<oon <3<sano<soasu<oaa ,pcfiuusoooawnaofiésa,‘ m<meu wEmzom=o H H.883 a H H H H N H H oNNmm-< $83 omHom-< OMHmm ONNmm Nmem vamm NNHmm vaom mar—LL07? wmcmmzé < H.m3mm-< H.Nm~wm-< < < H.vamm-< < < H.833 H.383 < 805—58 52833. 3533 .823 .3550an HEP—8 he 3:3 82 e SE95 8:33...“ Hufluz<3<u<m3<<§<<<soo Hoflusu<m<<wo§o<o§<<ou FEES/683033838 FEE<<<UB<<UB<<038<U 33 Htmhu<33333u30<us<0333<3 Sfl2<sa<<8<<8<<uo<u htmhu<uaz<<33<u<wwa<uuso HbmHU3303<suwsss<0<<us<< <3<=<o<<o<w<8 Eflu<<<u§<<33<<8<<8 Fuflu<<<8=<<38<<8<<8 €£v=<<oua<u<<u3<<3<u Sfln<8=<83<o==o=<<<<u €£2<§w<<u<8<<<=<s<u HBSS<<988<<8<U§G 85% . 353:0 H.m~Nmm.< 35692 8:8 E%%%%.H.Hm~mm HmHNom < < %%%E%%% .95 mmmmvé< $95-3 $39 3wa flammqé‘ dammvé‘ H.mHmmv H.vwmmv 38$ :25 Bank 9 3 o< Q< 333-3 353-2 o< 3 339-9 dmmwmvé‘ 883-9 PUthUU<<uuuoo<JUDUDDUD< HUmFUUxU/‘uunun HumkufivusunDD tomtu03<30<u3 OOD HEPUOD 3003 3:303:09}. hUmhuUDOD<<JD<<ODDmVD<OUD .HUmHUmV<3<u<<:UD DU$DD<O<=D< DUB/US“.

DD kUmPUGDoDUDDD<<<O<0<3<O< ._.Um._.UU<<uuu00<:UDUDDUD<3 DDD<GDDUD<<<00< HumFUDOD<3<<<uD<QDU<O<<< hUmHUDOD<3<<<uD<ODU<w<<< hbmhuw<<333030<<<u<003<3 hvmhuoOD<JU<QDDU®DD<0<3D DDD<u<<uD<OOD HEP—90.4:03<3<3330<JDJDU< hUmHUUUUDUUG§u3<uUDUDD< HUmHUUU<u<<<<O<Jw<<uUUD< HUmHUDDUDUODOODDUDDO<JO< 303300<333< ODDO<JDO<JDD<<JD<< fidmmoméx fimmfimméx Hdhfimmi H.wwaom-< H.w~H®m-< H.~mfimm-< ‘Hdwammé‘ HdHNmmLQ H.¢Nmom-< N.V~Nwm-< HdMHomLQ fimwfimmé‘ 35%: 338k 3:22 fimmaomé. Hdmmmm-< H.8Nmm-< m-< . _ €§=<o<<o<o<8uooo§wo puflu<=8<<u<<<<o<oososo Fumpu<ous<u<<u3<<3<u<o ankuz<<3§<<uw<<030<s<u Huflu<<<zws<ou<o<3303<=u H3533<33u333<<<0<u<u hvmhu<3<o<<0<w<suuooogw hamhusu0333<o<<us<<<<uuw hamhusssuso<u3<ogs<s<u< Fumhuzsauso<us<0333<s<u< Humhv<3<uu3w338<w<<<ssu hbmht<<83<<uw<<030<3<uu puflufiuiwsaoéifiéfi pufluaw<<u<8<<<=<=<u<8 Fufluséoéoéoaaogooo PUMHU3<OOOSDU<DUDDDDOJOO FEEE<8<<0<<8<5<0<< $fl€<<<§<<u<<<<o<oos FUmFUDS<<33U<<3U<<SU<<UO .éé . é; éa% H.33m-< gE gEgEH.mvHom-< HKhHmmi éx m-< m-< H.Hmfiwm-< EEEEEE EEEEEH.Nommv-o< H.0Hmmw-o< H.mmmmv-o< Héowmvéxx HUmPUODDOO<sD hcmhvuo<z<u<<sunwua<u<33 bumtuoowDODUD D<uu<333<3< DD<<<0<0<=< hanUUD<<<uu<u<<<<®<30<< HUmhuwDUDUOODDDDxxo: hUth<OOD<uUDOD<<3 DUD<< hUmHUDDD<ODDUD<<<uO<3<u< HUmkuwD<uUDOD<<33<<ODDOD hUmPUUD<<<ow<3<u<<JUD HbmkUwDDODuOD/Euxxonb 003 $fl8330<38<3<38<=o3 5%08353830833 hvmhu®<<U<<JDDDQ<0<JDDO< PUmHUD<<<uD<ODU<O<<<<<3< 342033 HumhnoDwDD<<JDUO<OO<<0<< HUmHUODDOD<3<<<uD<0DU<0< Hbmhuo<003<su<u330033<o< HUmHUUOGDDD<<DD<uDGD<J<< 3(33 .HUwHUUUDDD/an0943300939».

H.No~mm-< HdNHmqu .H.omaom-< Hémammé. HNHNmmLQ fiwfimwmé. fi.wm$m-< fivNHmqu H.N¢Hmm-< Honammé. H.0mem-< H.mN~mm-< 338-4. H.333 < H.330: H.883 H.383. H.~m8m-< puflu=<a<<<soos<<<38<<u <303<Ou<o<3303<3u0 hUmHU3<su3u333<<<O<u<uuu PUmHUJJU<3933330300333<0 hUmHuss<u<<u3<<<<uuo<o<u HUmkU303<390333<0<<u3<<< HUwHU<u<<u33<<33<u<003<u hUmhu<Ou<O<ssoz<zuODg<0 vahu<u<3u<<<s<3<u<3u<<o vahu<<<u<us<003<o<3<<00 Hamhusu<<<sozu<<<<33033 Huflg<zssszuso<us<0333< HUmHu<<UJJ<<33<u<OOJ<uu3 Hamhusauszuuzuw<<33<<u<u u3o<uz<osss<3<u<<u u3§u0<<030<s<uu3u hnmhu33<3<u<03<<33<<<uuo HUmhu<<3<<<uzo<s<uw<u<<u HEP—vaguugwu’xusxxéoo %E % gggggggggEgg% EEEaEEEEEEEEE bu<<uuuaon<uu<o<<3 PUmHUUDOD<uU<w<<JDDUDO<< kUmhu<u<uUDUDDDDODDGO<33 HUmHUU<uUDUDDDDGDDOO<sDD PUmPvODDOD<OUDD<JUDSUD< ODDD<<<33330UODD<3 baboon: ., HUmHUwODDGDUOD<Ju<ODDD<3 DD<033UD<<<UO<3< HUmPUDUDUODJDDD<QJDUD<<< . .owqu: H.NMHmm-< H.¢MHmm-< H.wm~wm-< H.oo~mm-< H.0mHmm-< H.¢hHmm-< H.wommm-< H.0HNmm-< ~.mvNoN-< 882529wa HUmhu<33u3003<u<000330<u su<0<<<33u3003<u<0 ._.Um._.v<<3uu<<u<<<<0<00303 Hnmhv<<<zuu<<u<<<<0<®030 PUmpu<3<<<uzmu<3<uw<u<<uu HUwHU3<OOJ<0<3<<Uu3<u<<u Hbmhn<3<<uwo<<<<333<<<uo Pbmht3<su0333<0<<ua<<<<u HUWHU333<0<<u3<<<<uu0<O< 3: TEBEGJN H.HmHom H.mmHom H H H H.mNHmm H H H “3 nmwmm mmHmm mmHom-< mowmm mHNmm NVNwN < < < < < < < < 82820:: H.wmwmv-o< Hfimwmvéxx H.0mmmv-o< H.howmv-o< Hdnwmvéx‘ H.mmmmv-o< H.Hmwmw-o< HéHmmvéd. .H.wmme-Q< 88533 Table 4. Results of single dose screen using ANGPTL3 dsRNA sequences The experiments were ted using modified oligonucleotide duplexes listed in Table 3. The sequence ofAD-15838.2 is identical to the sequence of AD-l 5838.1.

Delivery of siRNA duplexes was done using LNPS.

AD 15838 2— - WEE—— -m-—— —m--_ —-—-m —m-—— AD-45882.1- 0.95 AD-45883.1 0.98 AD-45885.1 0.91 AD—45887.1 0.35 AD-45888.1 0.80 89.1 0.91 AD-45891.1m 0.82 AD-45892.1 1.0'9 AD—45894.1 m 0.59 A0-4589_5.1 1.00 A0-45898.1- 1.09 AD-45899.1 m 0.59 00.1 m 0.80 AD-45902.1 1.03 AD-4590311 1.02 AD-45904.1 0.87 AD-45909.1 m 0.73 AD-45910.1- 1.01 AD-45915.1 m 0.48 AD—45919.1 0.98 AD-45920.1 1.00 AD-45924.1 m 0.67 AD-45925.1 0.008 0.100 —m-_— —m-—- Table 5. Dose response screen reSults for ANGPTL3 dsRNA sequences The experiments were conducted using modified oligonucleotide duplexes listed in Table 3. The sequence 5838.2 is identical to the sequence ofAD-15838.1.

- Hep3B leo lcso . 'cso weighted ' weighted leo I (nM) IC50 ll (nM) (nM) leo l (nM) IC50 ll (nM) mm :52 Nxmmeg Om 853% 0w 2? i=8 58 83?: 033.

E x09: <Z-mc 3%: 9 m 3on3 3.532 >3 w>< QELUZ< ”33> w>< coE—EE 028202530 Xmm w>< @3355 95: BEBE i £588 3% win: b:5m..> =EE>=8 $8338 Awmwm .3 053 TQ< 3.33— zuquCogxo mo .e 8:263 Bank 2F 2w mmdw mmNm Om oméN Om Hmdm Om owdm EIII'EII ll o w>< m~.HoH 3% w>< HgfifiaaagHaaafigfiHHHEEEEHafiaaaaHafifiaaaafififiaaaHHEEEEEHafiafiafi mafia IEa I w>< mg as 3: 3m 3: mg mg Hafiafiaaa .vm.mw § as as 32% hdeH afiwm mmfim as :8 Em moav :2 3.: :3 ”E 33 :3 :3 3 2.: a3 oo~m7o< _ob:09 EE EE $9.2.

Ai EaEa5%5%5%5EEa5EEa wflm EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEOEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEIIIIEIIIIEEIIIIEIIIIEEEEEEEEEEEEEEEEEEEEEE8N H.wnwmv-o< é m._._.aoz< Ea: 2%: 23,: 2% 253. .25 2 .255 23% .223 BEE .25 age .223 : 2 : E m: EEEE0mamam EHHEH m ,w>< mix: 3%: m>< 38H Eaw>< afiaagafiaHafiagafiaafififiaaaaaEifiaaaaaaafiaafiagfia 05mm 8.3. w>< mam Em 2.: as 8m Eaafiifiaaafi 91; NEE: $3-2 Hdmwmvé< H.vmmmv-o< H.322 322-2 233-3 9%: 25,: < mdhwz< m._.E.puz< mAHaOZd. mfiuoz< HHHEH $32.8 EH! HE! 2%aHHHEH 8m: omem 88 83 2% 33w $2 fig NS: 8.? fig as ”.22 m~.mm 8a a; 2.8 2.3. 2.2 a: H.mNmmv..o< 339-2 8322 85-2 32-2 IIIEH m.:.a®z< 3572 6581+. E .252; 3:8 EEHEEE No.8 Ea 8.8 8.8 8.8 8.8 8.: 8.8 8.8 9. .288 8 8.8 8.8 8.: 3.8 8.: 8.8 8+ .88. 3 cm 8 8.8 8.8 a 8.8 EE%E R8 .88 E 8.8 8.8 8.8 8.8 8.8 88 8 8.8 E 8.: EEa 8.: E 538 8 a w>< 08 888 E 8.8 8.08 8&8 :88 «38 8.8 >3 HEEEEEEE 8%: o>< .288 88 888 8.8 8.8 888 BN8 8.8 888 2.8 8:8 8.8 w>< 5.88 88 8.8 88 8.08 84.8 3.8 3.8 3.8 8.8 8.8 .888 EEEfiE 8 8 8.8 RS 8.8: 8.88. 8.8 8.8 8.8 8.8 8.? 8.8 8.8 8 8 8.8 8.8 R8 3.8 8.: 8.8 8.8 8.8 8.8 8.8 8.8 . EEEEEEEEEE 888-3 -o< 388-3, 8.8.88-3. 388-9. 8.8822 388.3 388-3 388.2 8882‘. 8.88:? 388.3 H.232: 398 mfiawz< 3E02< mfiauz< mfiauz< BEoz< mfiqwz< mfiaoz< mfiaoz< «.863. flEoz< flEoz< «.863 2%. .vw.m 9V2“ mega.

NmNm ofimm oo.mm 55:; :8 . action NfimVVHOIEZ won -won wném vaémm whém mN¢.Ho¢ vmoH-NHoH mnkm oww-wmw mHvam 09mm mmoHénoH momev hmfih oH £3-32 <03 363 85:63 2:333. 03 ‘mmwég Houcfimwnnm DUDwOODUODD wmcwmzct *0 DDO<GDD mOz 39.0 O<03 n: E 9.3 UUDDDUD¢DDODDDOD<<D<D<< D<<DDUO<0$<<G<<<<< <D<u<030<5330<w330<033 U<<w3033<<330w<0w<<0<<< <30<003<DU<UDDU$DD<0<DD QDGDDEDDUU<OG<<0<<<<<D DDDG<< 0<0<w330<<<< DDUD:<D<U<030<<UDDG<0.DD w<wU<<w<<<<<D< DDwD<<D<D<<UDD<D<<w<<<< 033w<330<330<<33<<03333 <<<<D<<U<<00<0<DU<<D<<< <603<DU<UDDUUDD<0<DD<<D 0<DOD<DU<UDDUODD<O<DJ<< uufluwzncoomeEU H H H H H H 3:335. mEmz H.mmmon-< H.HHmon-< H.HHVon-< nemon mvmon MHmon 8va < NvaoHLq < 4. < .movon-< H.mH.mon.< H.mhmon-< H.mmvon-< H.mmmon-< H.mmmon-< H.mmmon-< mAEUZ< ‘ :_ VmoH w~.-wm mm mmv mm 8m mm mm 098 nmfin be :2:on maggot—22 wwOH.omo.H. wifiwmm m9» vHoH 8w MHv.mmm wmoHénoH .069me hmoHAHoH mmoH-wHoH 805.58 we 98.5 8533 .332 EEO mo: 335.68% $.5ng 8:3 9 33 $3283me x33<3<33<u<<<u<<u<w<<< xDDDDDUDDUUDUw<<DD<<U< x<<UDU<<UDU<<ODDU<UDOD xDDDUDDUUDUO<<DD<<U<UD xDDU<<<<u3U<<UDU<<uDDU <<Uw<<wa<D<UUDU x<DDDDDUDDUUDUw<<DD<<U xDDDDwEUDUDU<<UO<UUU< x<<uau<<UDDU<UDGD<D<<O xD<DDDDDUDDUUDUO<<DD<< xDDDDUDD<D<<ODD<D<DD<U x<<<<UDD<<DDU<<DU<<DU< xDDD<DDG<DUDUUDDDDD<DD x<DD<<DUD<<UO<<w30<D<U x33<<3u3<<uw<<w30<3<uu % E HdeonLq H.meon-< H H H H 355 H.wmmon-< H.onon.< womon H.mH¢on.< NHmon n-< H.w8on-< -< Hdhmoné. wmvon NwaoH H.Nmmon 8535:: < 4. < < H.<mmon.< < H.mwomm-o< H.mwm~m.o< H.omomm-n_< H.mmm~m-0< H.H8mm Hdwomm H H H H H6N0mm .h 9.6.95 HthNm vwomm HdmmNm H.wm0mm o< o< HnmNmé/x Hnommé< o< o< o< < o< o< H.Nm0mm-o< mmeNm «wMHflwMH £8-22 $32 Hw.mm Neméww nmN mm mum mvm mm 5%%%E520:8 5 omoH-wuoH mmm 83% 5. «003463033 DDO<<OD 34033: DUU<<D<30<O<D<DD 38033333 5332588323333: 033<<DDUG<00<<0<<<<<D<< 330<336<<33<<0333 3UUUOODDD<<DD<UD$D<D<<< 0<<<<<D<<u<<u0<w<nu<<3< ongoo<3<<<u3<wau<o<<<<< uoaaw<w<oaa<<<<< 2<08u<8<83<83op<<33 885838333333: aaawnaogaugnoiaai 33303383352333 885388333383 33022235833 DUDQDDODDD¢D<<D<D<<UDD< 38333335038533: o<o<8<§<<<oo<oo833<< 8383833585235: H.Hmmon-< H.meon-< H H H H H . . meon HHHmmoH H.nmvon-< Hmmon mwmon mHmon mwaoH mvvmoH < < < -< < < MSWMNm VWMHéwMH mmoH-mHoH HwHo mom wmv Nwfi .thKvm mm-mh onmm - V a 555%5%5%EaEa5%EEEE3% H x<<<03<<UDDU<<<<U<UD<w x<<3<3030<3<3300<<003< x3<<303<<u0<<03w<3<003 x<<<<<303<00<0<3393<30 WSD<DDDDDUDDUUDUO<<DD< x<<<u33<<38<<8<<8<< x33 x: x3 D<D<U<03<<DD<<<UUGO x3<38<838303<3332 5233835333332 x33333o<<u88<<uu<u8 .x<<33<u¢wo:<83o§oo<u x<<3<3<u<8<<w<<w<w8< 53<<38<<8<<8<<uw<w 5<3<<<3uwn<<<8u<<u<< S<88§<a<o<<u<u<8u S<<u3<3<33<u<<<u<<u< w<<u<8<<<3<3<u< 3303 x<<3 w<UD<wD DD<D<U<<U. 3<<0<UUDUUDDD<DDG<DU Du<<DU<<DU<<Uw<G<< H.0mmon-< H.wwaoH-< H H H H ommon ovmon meon . wngH < < < EEa 38on EE5E5HéwaoH < < H.mnomm.o< m H H.HomNm-o< H.mwmwm-o< H H H H H mwommé nmomm mwmmmé H.monm H.m50mm Hémmwm Hdmmmm moomm o< < o< < mmmmmé< 5%EEE5%Ea DVHmmé< Q< H.momNm-o< o< o< o< o< wmén omv-wmv NmoHAKoH mNHH-moHH nmwfinw mwMH-mwMH wwméwm vaH-~hHH HmNH.mwNH 2?an mmmAHm oH mmNHHNNH nomhmw aon-\.on Nvmooww mwméwm NwHéwH oonnm 57mg» <<<<<3<<0<<00<0<30<<3<< 000<00<303 303 3 40003334030033 <03303<3<<<03<030<0<<<< 33(003300<<3<3 03 0003 033303<<3<3<<03 33 <3 <030<0<003033 30333 3 333<<0<3. 3<3< 300330<3< 0<0<3<33 3 0333<3<00<<< 3033303<<<00<0<<< 303300<33 35533533883333 38503683333533 oaaoaonoo<<3<n<<u3<o<< 35533328338533 53<08<o<<<<<363<u<3< anuaoasuaaaoafiogéga 38383828533223: 330<030330330<303<3<333 <<<00<3<0<<303003<0<333 03030333<<<0<0<3< <30<<0330333330<0333<3< H.H~mon-< H H H H H H H H .mmgoH.< .hwmon.< .mnmmoHi .nnmonLq .< .< .mmmon..< .HwaoH.< 5EaEEaEH.mmmmoH-< Hfimmoni Hdwvoné. n-< omvémv ~8H-NnoH mNHH-moHH nmm$n~ mwMH-mmMH wmmévm vaH HmNH mmén m3 m8 $8 wwm NwH ooh HDV whHH HNNH mam m3 $2 83-22 8*”-an 32.38 szmm wvm NmH owm Hmw 0<3030033033<333 x<3033<<<<<<0<<0<30030 x3<3<<033<3<33<0<<<0<< <00<<<00<03<<¢00 x33 x33 3030<.03<033 303003 x<<<3 3<3<0<< x<<<3<3030<3<3300<<003 x333003<3<<<0<30<<0<00 33<0<<<0<<<<0 x<<300<<0<<<<0<003030< x<<<u8<<u8<<w3853o x3<<UDB<<<<u<ua<uoa<o x33<3<<o3<3<35<<6< S<<<8u<<u<<<<o<ooaos 5<Ba<u3<338852< 3533333555220 x<<<<3<<<uau<s<uw5<<u x<<<3<3<0<30<<0<<0<030 03(00<0<3303<3003 x3<3030333<<<0<0<000<< x3<3<<<030<<<<<0<<0330 HdeonLq H.w~¢on.< H.wmmon.< H.whmon.< Hdnmoné H.ommon.< H.0mmon-< Hémmoné. H.onon-< EaEa5%Ea5%5%Ea H.Nmmon-< H.wmmon-< H.ww¢on.< H.wmvon.< H.mmmNm-o< H.nmomm.n_< HNwommé< Hdfiomméa. H.mhm~m-o< m-D< H.Hmomm-o< HéNHmmé< H.mNHmm.o< H.whmmm.o< H.mmmNm-o< H.mNomm.n_< H.o.Nomm.o< 08»-me wvméwm www.mmN mwvfivv mmw-nmw hvafiNVH me-mmm mhméhm Hom-mDV meHwH wHNH.omHH HwH-mmH vwm.~vm mmHkmnH mmoHHHoH Nnvémw ONoH-wmm «OMH-NwNH wNHHéOHH monwa <<UDDO<ODDO<wDDDD DUUD<DU<wDDD<DDDDUDDD<D <<03.DD (065 DDQ<033GD<D<<<UD< 04363 UDDD< wDUDw003u0330<0<0330<<< w<<<UDD<UDDO<UDUUODDD<< OD<D<DDDO<DODDU<GDDDDD< DDDDDGDDUD<DD<DUOD<ODDD DUQDDG<DDO<DD$<<DD<<ODD <<uo<3<u<<33835<3333 03033833: 3<<<uo<3<u<<83835<3 8235838333333: nou3<u<3323<<<<8woaa< 3338386383333 33385333? 8338383333833 30233333833333 83330283383334: auooao333<<<w<o§<o<< H.mvaoH-< H.mw<on-< H H .meoni i HNmeoH‘é‘ H.mmwon-< H.movon-< H.mm¢on‘< H.mmvw,oH-< mwvég th$NvH meme 5%H.mmmw8-<. $9w~m wwméom www.mmw wnmémm Homev 5% $32 5%EaasEa5%as5%5% x<<<<UDU<<UDU<<wD DU<UD x<3<<<0<<<<3<<<03043<u xD<ODDD<D<U<<UDU<<<<<U x3<<<w30<<<<<u<<u330<3 xDDDw<<UDUDU<<Uw<UUU<0 xDD<<<Uw0<wDU<<OD<<ODD x3<<<<<UDw<<U<DU<<<D<D x<<<UD<UO<D<<D<O<<U<<< x<<UDD<<DDU<<DU<<DU<<U x<<<<3o3<8<u<32o3<8o é<<<uw<<<u<uz<uooaa< x<<3o3<8<u<302<3o32 x<3<3<u<8<<u<<o<o8<< 3<<8u<33<<<<<83<o x<83<<uo<<w3o<3<uu8< <u2u<<<<<u<<u3 x<3<88<3<3<o<<w<o<8 x<<2<3<<<aou3<<<8u<<u 23858553830 58388333<<<o<u<8 H.<vaoH-<. H.Nw¢on.< H H H H H onmon 33»on wmfion £3on vovon < < < < < H.vw<on-< H.vmwon-< aEaE H.~3mm-o< H.HHOmm.Q< HKmmNmé< H.woomm-o< H.mmomm-o< m-o< H.NHOmm-n_< H.voomm-o< H.HNomm.o< H.mmm~m-o< $23.3 333.3 gamma: 5%Ea555%Ea5%5% HhH-m<H me-mmw $3: $13 233 23mm Hawmmm 25.2% $38 38-3: 23% 33mm. $33 :23 32-88 533 33: mmmém NmfiéRH 33R wDDDD<wDDUD<<<Uw<D<U<<D UUDwD<<D D<<03303<w03 UDU<U<UUDUDDD D<D 0<GQDUDUGODDDD<QDDUD<<< <<o<3o<<u3o3338533 <3<<o33o3<83< 3<8303<<3<<o33wa<83 03838533333833 asu<w<038<<<<<w3<u<u<o DOD<OUDD<DUD<UUD<®DGDDD DDD$D<<D<D<<UDD<D<<0<<< OD<<DD<<WDDOD<wUDD<DUD< 38333803033838? 33<usu3<3<<<w333<u3 833%:5832333 3383333828533 83334283883333: usouo<u<333<<<<8833 33<<w33w2<83<83<83< 30.330w<333 30<GDDGD<D<<<UJ<ODU<G<< n.< H.mHmon-< H.0NMwOH-< aEEEa H.mwaOH-< H.mmmon.< H.HwaoH-< HNHHmH me-wmw. NwH.N¢H $33 53% 33% 33% 8?me Hmm hmoH HNm HHm whoH Hom 5%5%5E5%5%Eaa?E55% x<33GD<DUwDDD<0<<UD<<< x<D<<0UD<U<<UDD<<DD<U< x3 DD<0<<UD<<<<UUQ<G<UU x<<<w30<<<<<u<<UDDU<DU x3<<oun<u<<UDD<<DD<U<0 x<<82<u<<u3<§=<u<uo x3<<<w<<<§<<<uow<3<uo 28303533333388 x<<<u<u3<003<w<3<<003< xDDDUDD<D<<ODD<D<DD<U< x2<0<3<<003<u<<033<<33 o<<<u3<uw<a<<p<w 3532533333653 x<<<u33<3<u<u3<<33<<< x<<uzu<3<88<3<3<o<<w x<<8<<uw<u<<o§3§<< x<<8w§33<<<<§oa<8 S<oo3<w<a<<83<u<<u3 <<u<<<<o<ooaopw 58853533325: Hdmmon-< H.meon-< H.wwaoH-< H.vaon.< H.meon.< H.<Hmon-< H.0wwon-< Somme: H «waoH < n-< H.0waoH.< 3882 3383. H8803. dimmed,‘ H.N3Von-<‘ 2&8: v< 2383 :56: H.wnmNm.D< H.¢w0mm-o< H.Nnmmm-o< H.N8mm-o< H.wnomm-o< H H H H NuOmm 38mm Nonm 38mm monm Nmomm :ommm 38mm 32mm D< H.moomm-o< H.08mm.o< 3. o4. o< 3 #3293 figmmmé‘ H8393 D< 3 3 3 38%.? moméww nmvfihv @515: mmmfimnma Hmvémv nnmymmm Nwméwm. ovméfio mmwfiaw m3.m~w www.mvn vom-~wv mva -vaH Nm¢aémvH aomaéhma momaéwma Nwmévm omvH-wNvH “Kean—”mo...” mmHHAVHH .<D<GDUU<DU<GOD<UUDGD<<D <D<<<DUDDUDUODDG<DDO<DD DO<D¢D<D<3330<DGDDU<ODD D<UUD<03033330<<GDD<UDD unnuauonpu<33w<3w<§3< 33383380385322: «3383838332333: (<03 <0DDG<UDDDDU<<UD D <D<DO<O<D<DDDD<ODDUODDD DDUDU03<DU<ODDD<DDDDUDD DSDDD<DDUDDD<U<DDDDGD< 33333333535333 83533588333533: 33523588332653: 03333833352333: 3338533523333: UDDDGU<UUUDUDDU<DGDDD<D <u3<u3u<u3uonao<<<3 U30<<UUDDunw<UUDDDUDGDD DO<UUGDD<U<GQOODD<UGDD< H Hfiwvwofié‘ é. fimmmoni 3383 H.238: 3383 H.238-< 3883 :38: H838: :38: H.3mwofi.< $383 Hdmwov,‘ #2982 Hmmmwofi 3S8: :38; H .momon -4. < mwmwofl-< Hmwénv hnmkmm m E%E%E%%EEaaa%VaEE D<U<003<UUD 0<Dow<u3 x<<DU<<DU<<U0<0<<O<DDD x<<UDG<<U<DU<<<D<D<U<D x<<03<<UDDU<<<<U<UD<w0 S<§8<<8<<8<<uw<u< x<<a<<<u3o§<uo<u<<uu< x§<<<u3u<3<uo<u<<8<< <<<<u8<<u8<<w x<<<8<<ua<<<<3<88<3 x<<o<<<<2<<<uao§<uu<u <§os<<<w<§<<<u 5<<<<u3<§3<<8<<8 x<<<33<<<uuo<u8<<oa< x<<<<333<<<uow<w8<<os x<2<<<3oun<<<8u<<u<<< <<3oop<<<8u<<u< x<a<<<u<8<<o<oooa8<< x<<33<<<uww<w8<<wa<< x<<u<o<<<ooau<u<<ooaao x3<<uw3<<uuuunwa<<uwwb H.v0mon-< H.w3on-< H.mmmon-< Hflwwmoné. WE5aa 39.83 H H Nmmon 3%on < < 3383 3383 dmwomméx‘ H.v3mm-o< fiwwmmméxx H.5m0mm-o< H.m8mm-o< H2893 28mm fimnoMm 32mm- S83. 2 533.3 EE5%5%555%EaH.m3mm-o< H.MMHmm.o< o< D< o< moméww mnmfimm 32$: mHNHémHH 33m: $3-2m 8?me 83.53 83R mmHH-mSH owHH$HH HmoH.$oH mmHHHDH 82.3: 33,5; NHmémw Slow m--mo~ mmoHioH mmméHm 03uu<3u<003<00303<<33<< <DQD<D<DDDO<DGDDU<QDDDD 00336690003 (43.03 UUDDD DD 3603 <<UDD<UD waUDD 333033 DG<UDUUOD DUD<<< <<w<30<<UUUD<GDGDDDUGDD 30<UDUUODDD<<< DGDUGD<DU<ODDD<DDDDUDDD D<3<<<DUDDUDUODDG<DD UDODDGDDDOD<<D<D<<UD U<GQD<UUDQD<<DD<<GDD o<3 SDUUODDD<<<DDDDUUwDD<D <<<UD<ODU<O<<<<<D DGDDDUD<<<UU<U<<<< <00:D<wwOUUDDDDwDDDUD<< D<D DDODDDUD<<<UU<U<< D<<<UU<U<<<<O<D DD<<<D 03< 5330<033w<633330<<0333 <<ODU<<UUUDmuD<UU<w<<DDD ODDDOD<<3<D<<UDD<D<<0<< DDDOGDUSDDDFGDDDUDDUDD H H.Hovon-< H H H H.mnvon-< H H H H H.mwmon-< H H.839: H.mmmon-< H.m3on-< H-< H H H .momon mowon meon nmmon mwmon HvaoH Hmmon Hnmon .4. .Hmmon-< .mowmoH-< < mSVon-< < < < < < < < H.5mon.<. woméww mnmémm thHRoHH wHNH-meH wSVH.wN¢H wvwéNm 09 mm: mam mm: meH HSH mmHHéHH meH.wnHH GSVH-mN¢H 238 5 mNN meH wmm as mm: E mHHH 8.: :8 By mom 33 on x3 x<<<<UDO<<u<DU<<<D<D<U x3 <<U<UD<GwODDU<DU x333<<<000<030<<03<<03 <<<3<<<030<3<uw< x<DU<<DU<<UG<0<<w<DDD< x<D<<U00<<<<DDD<<<u0w< x4533 x: 5 x<3<<023<2<3<u<<<u<5 Sposooaps<o<<<u<<<<oo x<303333030033 x<DDD<<<UwU<wDU<<OD<<0 xD<U<<UDD<<DD<U<0va<UU x<<<wDDU<<<<Uau<<UDU<< x<<<JDUDODD<U<wGODDO<U x: D<<DD<U<GOD<UUDO<D 3 3336: 3303 DUDD<D<<ODD<D<D 00 u<<<<wwuuua<< DDD<D<U ww333<0<<<u<<<< 3332380835 3<0<<<U< D<U<< x<<0<<0<<<03<<<93w<uu< .wOm on ‘HNmmmoH -< E aEaH a Nwmon.< aa < H.wmmmoH-<_ EE E H.Honm-o< H.ooomm.o< H.NHHmm.Q< H.~.onm-o< H‘HNHmmé/H. H.w3mm-o< H.wm0mm-n_< Hde HdMH mm.o< HmeNmé< mmé< H.onmm-o< H.Hwomm-o< H.wHHmm.o< H.mMHmm.o< 323-3 Hhmwomm 3. H.wmmmm-o< a%.5EéEHmméww émma-.~nmfi .

.HmvH (Hmwlmmwa %%E%aEé% .wafl (HNUIvmvH ww~é¢~ 883833: 338888333383 38323332883323 55358353333832: <<ua<u3<o<<<<§<u3<o§ <5338oo<oo<838333 D<DU<GDDD<DDDDUDDD<DUDD <<3<DO<0<D<DDDD<DDDOGDD 3<UDDO<UDUUODDD<<<DDDDD <w83wwo8u33u<o<038<< 8333338385333: 3533385383833: 0388333333338? unaua<<<uo<3<u<<83oua< anuasuwwausuasaiéég 33353833335333: 232838833353 <UDDG<UDUU$DDD<<<DDDDUD DDUUUOwDDD<<DD<UDGD<D<< EaEaEa.5%%%Ea5%a5 H.Hmoon.< 5%EH5%5%a? fié‘ H.mwmon.< mmvfl-mm3 vmvHémvH ww~.wv~ x<3<2<o<<o<w<8uooo3o S<w<<ua<<<<8w<o<88 x<<3<<uwo<<<<33<<<uow x<<<uw<<<u<un<oow38<3 x<83<o2<33383o<§<o x<<<<<o<<u§88uu<<<o x<<w<3<<<u<<<<3<<<uso< x<<8<<u3<<<<3<38<3< x<<<<<DDD<<<uow<wDU<<0 533683253823 uoo<<u<8<<§<3< x<<<<o<<w<8u8uo<<<u= 5382235358 352333583233 5583 332 253253558532 x<0<<<<333<<<00w<030<< x33<3<u<03<<33<<<uuw00 aE33on H.w8on-< H.Nwmwo.n-< H.mN.von-< Héwwwofii 2%on H.0mmon.< 3S8: H838-< 39.83 HNmmonLq H.Nwmwo.n-< $83.3 H.$m~m-o< 333.3 fimgmméd. fimmmmméd. H.Hm0mm-o< HKHommé< 333-3 H63” mm.o< H.mmom.m-o< H.m8mm-o< 383.2 E5a?5%5%5% H.0Hamm-.n_< flowmmméa.

NNvHéog HON—YEAH mNMHémmH , .mmvH mvH «NHH-NOHH 0mm-wo~ NHMHémNH 33-me NHNHémaH Nnmémm mmmfiflg www-mmm hmmfiémma Nmméhm Hm~-mo~ wmvHéMVH wMNéHN mmvfiémg mmSTmmw: .<U3<0G3<0GDDODUD<<ODUDD 03: JUD<<<UU<U<<<<0<DO<< DDU<0<GDDDD<UUDDUU<<D<D UDDOSDUUODDD<<<DDDDUUD <<UUUDOD<UU<w<<D <UU<0<<DD 0w333<<<3 3030423 DDD<UU<DDD<3<DDODDDOODD <<UUUDDD<ODGUDDDGODDw<D DDOGDDwDUwD<DU<wDDD<DDD <u<<<<0<3u<<uu03<03w333 UDDU330<DOD<D<DDDO<DODD DQDUDU<D<U<U<DDDDD<0<U< UDDDDDQDDUD<DD<DUUD<QDD DUDU<D<U<U<DDDDD<w<U<DD 3853335388333 <u8303<8<u<<3383w<<< 3883333333883 3U30<<<U<w03<3 2.50wa D<uwDD<GOGUUDDDDODDDUD< i fiaowwofixx HNNwwowi . Hmemoni. H.mmmwofl-< .Hthoni é‘ H.mn¢wo.n-< H.mowon-< 3283 $883 3383 3883 :88: H.mmmon.< 3383 3283 H.3mwofl-< 3383 NN3-NowH Homaéw: wnmaémma mmVHhmva 0mm NHNH H3 OHN NHmH-NmNH vmgéo: HNm mm: , %a%EaaaaEE 33<w<u<<uua<uun< x: 3935: 30300: 33<0<< x<J<DDOG<<003<<<<UDU3w x<0w<<<<333<<<uww<030< xDDu<0<<<DDUDOOD<U<DGO x<<uu<<<u<<3<3<<<3003< x<DU<<uu<<<0U<UD<<<000 x<<<3<<<UDu<D<uo<u<<UU x<<<U<UD<QOODDU<DUDDDD x<<u<8<<§§<u<8<<u< x833<<<<<2u=on<3u<o< x<<ua<uo<2<§<o<<u<<<< x<<3083<<<<<3083<3w< x<<<2oo<o<<w3<<<<53o< <<<283uw3<u<ou S<<uou<<<<33<<<uww<o 2388338552388 éouw<<<<33<<<uoo<u3 u<<<<w883<<uu fiwmwoni Hdomon-< H.0waoH.-< H.vmmon.< H.Nmmon-< .H.¢Nwwofi-< Hdnmoni H.Nnvon-< H.Nooon-< aaa -D< HA3 mm-o< fiwmflmméxu Hdimméd. Hfimmmméa. HdNHmmé< Hdnommé,‘ fiwwomméd. fidvfimmfiz dmwmmmé‘ H.239? 33%.? EE555%Ea,5%EEEa anHhmvH E5E H37mHv wmmH-meH -NHmH‘ DHNOIQmmH E%EE% DW<UDUU0333<<<DDDDUUODD 3<<o<ao<<oaso33333<uaa 32355533333233: 83350338083383: DDG<<UDDDUGO<00<DUDDUDD UDU<D<U<U<DD DDD<G<U<DD< ODODUDU<D<U<U<DDDDD<O<< 8323233333053 u<<w8<<uuuaon<uu<o<§o 3<uu<o<<3383o<<<u<ou3< 8<o<<383u<<<u<ooa<3 <<u83ws<8<o<§ H.mmoon.< 5EEa5% H H H MNwon hwwon -< mnwonLq < nmeKmvH HE‘HNV wmmH.meH EEa5%Ea5%5% 033.250 x<<uoo<<<<333<<<uwo<03 o<<<<<u<<u:3<82 x<2<<3u83<<<<<833<3 x<<<u<<<<oou83<<uo3<< x<<o<<w<3u8uo<<<o33 x3<<303UD<<<<<D “€230 :8 0394450 <<<<30303<36<w<u x<3u83<<<<§oooa<a<o x<<3uaooa<u<ooo3o53 S<83w38<w<<<383oo 25580385338: 23838353832953: 8:263 .H.wmoon.< EE5EE H HS: womon < 5aa-< 55 8:865 H.wNH nu mm-o< 333-3 3.23-9 383-9 383.3 $23-3 m-D< 323-9 #3399. H.~mm~m.o< dimNmAZ H.m8~m-o< 395$ '167 wucwsumm .>_w>_tw%e «5585 .832» Amucfimwnnm .9.5.2 we mmwwmwwnmwnuzusams no: EEO 992me MmquEDF—DJGED38,35 33 8waDEUEGBDMJEDED£<3<3<m $63635a<339m£ofl<£o£<2<m floeoefis308:5MEWS: fi<§o£ossgaseoeoggefi ma:”583533363035332m 9.5mmzubgeusssufia:£033: 55zbummfigfifizga 383630533gnawgssuzmtm 3<3<£<msfl<mb=5m¥<m5 3 :flomssES:Dubwmmmzfloflxxmzmw:m ug£<H¢<3<mu33~53<ww<fl<m 933335: mmt.)8.4E<8U$<wm$mum5£<m5£<m 3<MW<ED£<J EE»:?8¢~ZEU wmcwmzc< wEmZou=o HdmmwofiéH H.HHm8H-< .E H.Homon-< EaEE H.movmofi-< fimfimmodé‘ fimnmmoaé H.mmvaH-< fimwmwofi. fimmmwofié. mafia—02¢. 5 mod mod mad $32588. <30 022:5 ho 3533mm Awucemmnnm 3323235 DEDEDuS3582808435£<£< EUR—<83 80:35am UEUNEMS mmcwm £238 ,6 QUE/$30 9:23 32 .230 a. 035353330me£3qu3me5 853:5 cum. £<8<3<£< 8350359283 m5 «.25385588522358623: H55augeé3226:6335H6 85385$5635055:255 85:5a:3035556ng32.3 wasa:36.3553abzmbmzubuz 38<UOEOE<E<EO 03583333583EUCSfiOmzssmw/w 09535353533330532322580 <mUED£<328<m58<m58< :23: £<3533me533030memuO 95 wmcum 3:8 mEmZom=o H.wmmonp< H.onm.oH-< H.OvaoH-< 5%EaEaas5% fiwovwofii H.¢Hmon-< HénmmoHLq auq HNNmmonA H.vmmmofi-,< :52 a. H H H H x295 80mm $53 08mm «.mmomméaH fiwmomm fivoomm 363 .quuE 2 2 HémomméaH .H.Rmmm-0< 2 D< Q< o< uefiaoszeug:852:ng3% mumps£030353350:5835: 3633236303as:a 36509:fiauzusauw53<fl<33m afizms3335235:5: ”“5323: eu9<mm£<w2e§<2<m uaossmzezmzs3352:25353 35859953<§E<u5m5m5£<m mag£<£<a<mb330302328:m ma:mam3<3<e<u=£mausoefizm E3@meawasmmmzmssbgefi 3.2mm5sausagmmefib96:23:3 85“30553836ng233:: asweaaoeamEsszazuEe:32m aSSSMS3553:2533a:23m 363658858323236303:m mafia:36323585ausbaomzm 3:ssmsgeézeuezm 3236:2523:333935.295: H.mmmwoa-< H.HmmwOH-< 0H-< H.Hmmwofi-< H.vawOH-< HKHmwoaé‘ H.hmvmoH-< H.m0mwOH-< H.mfimwOH-< Hdhmwofia‘ H.Homon-< HHomwOH-< H.HmmwOH-< H.mmvwo.n-< fimfiwmofié‘ H.vawoa-< fié‘ dmmmmoaé‘ H.mmmw0H-< 95 .03? 0328 035 omd<£< 035 033 035 8:: 095 035 mad: mod 3 035 3.48383 03?. ~23: D :5 3< m5u5£<3<55 £6.30 m5 :5 m5 35 £435 03:33.8 348 £08 3 D Us «.333 mw<u5 938: E< :5 £0 30 386. 80355030me "wk/«30333020083 £<£00<m<m5wu<3<80 EU :38: £093 mzfl D ”280 €00<w<8<£<u5 2 E my DUB £03553 3:350? 055 DUE/”‘33 EE$<EU£<=U £<30¥$<30 mw<£Dw~03<~<~<208<mb 3<€<32<U£<£0 ww0£<m003< 3095 303230 8020me 3? P2. 34.360090 @953:302355553:=22:a: 8d:a:385335253532352 :5a:”253555£36385 wad/E:32023938858:n65 £5<w0fl<m¥<m58< £<£0m¢0 m3 £0 34.316230 3481‘ 200<£<3<3<£< Nmmwoa-< aEEa ofi-< Héomwofiéx H.meon-< fiwhmwofié‘ aaE EEdimwofii dwmmwofi< H.~momm-o< H.mnomm-o< 3.38-3 Smommé‘ H.880? m-o< H2893 H.829? $33-3 H.me~m-o< Hfihommé< m-o< donmwméxx H.m.nomm-o< HKvamé< H.moamm-o< H.mommm-o< H.m\.omm-o< Hdmmwméxx 1 69 mgmfiébgefimflo£3852% 3635886:5mmesflésmzmsa $2323:e§<mm8<fi<eu£<3<m Umwwmmzmwofiouh:3:535 ”£035 :mSmwzuSuS :5fi<3<: ::5m5mm3452m535m5m $53535B<m5m§530m53083m 8585MBm58<83<m58<9<£<m as2285eue<§eze<9<e<eas maumausbeamzsme:3<e§u£<m 33303:33<£<£U£Umw<m mmwumzwuflufiuflnusanEOEDEGmfl—D3 afl<m8<m5mw<mbz33353030353 signs»:353%532 3330:5532ng3553225 $855885ageoazfiogesm amUmE<m5£<mw<mm93:28:£0thm 35853:3033334658: $.35: :flomeghoeuga«82333583:5: 303336.303232353 H.m~mwofi-< $383 :Nmmoz fimmvwofié‘ Hfimmwofié. H H .mmmwoa.< dkmwoz. H.830: Emmwofi-< .mmmmOH-< Hafiwwoaé. H.mH¢wOH-< HHwaoH-< flmmmmofiix H.mHmwOH-< H.mwmwo.n Hémmwoaé. 35qu fimmmwoaé 035 omd< amt: @3230 omd 3 mad: mmd< omd: mmd: 035 $35 035 035 m5 @92me 3230809538 m5 :5 m2 £4.

EU E 3383 D 8U @7395 3.4:: £3 m2 PEPSMEJUEUUE £< ~3th .mmd:m58<muOuE£<GU£UE<£<30 EUQOEUEEQD 3235 35303232330230 359336 mmd<£<u5£< 0928,0358: omd<2<m5 EU 3< 30 33 8U £035 33 «”285 35 £253 E5 3 DC 03803 :5 02 :5 30333555 £353 3 mh<£<£<w<5m¥0m5 3523me mtg/«5233 33359330 fl<80202< £3 «5 £U£<£U EU flaws 3<£< m5 EU $<80£0M5 39635 mg £0me EOE/«flows £<mw<mww 360234.30 m2 35 2w £40: 93 30358033 £3358<£<8<£U£0 3(803953SUE/.3280 omd<£<3<3<£<38$<mw<$wfloEU Emma < aEaH.womwoa-< fid‘ aEEaaEEg 0H-< Hdmmmofixx Hfivwog‘ H.Nmmwofi.< EEEE EEEEEEEEEEEEEE mmzmeumzefizmas£35235a EUSSMGMSMSSES£<£u£oeaw mmsmmzmwwswzmw:5535028.). mfl<m303<ubus 3:328me 3&me3 ~96me 33353me 35$:me $3035 23235 9033 EDED‘m 353530355smfloiswzms:5: :mbmmsmsuzms3853553532: mefimse:eogwsegzafifism maomeamzawefise:382.053? ”£385mmwmwauwoqasmzmzmws 209 :Umzm mmw<m£<u53<u53mmbzbuw0353<m magma:m53553382333535m 3535303583«3323593£035: 38mm»:36333033034553.433: 35$qumem5£<3u30u5m5m5:5a D 3 0838033: acme:335essuepmzeoeéi Enasfl<mw<sbmb035m 3<m~<3 Ewan—Dszflufluflnszzzm :38: $382 H.mmvaH-< H.mfivwoa-< H.mmvaH-< H.ahmwo.n-< H.mm¢wofi-< H HmvaHLq OH-< H.movm8-< H.mmvwo.fi-< H.mmvwofi-< H.mmmwo._n-< Hdomwofié. fimmmwovd. fimwmwofié fimvmmoaé‘ -< Héiwwofikx mmd<e<303<3u£9<5202353: omdsmsususssNEEGWCNUUUNE @252:£5memessfibmzeaEu m9;$56.5NEUEUENGJSUZUS 032:2£32$825535:Eu 0353.0353235226eggs/‘5 8::~25ao£<£s<be§sEons mod: 0952<E<£Ouw<m55h<flo323035 035m2£896£52533:m5 833555E<3$<5£o£§<£< 838:5£38225923325 magefis325826235?£35 omdfifibngafiéz599592 032335382555333863: om332352235023o505$ wad:mwfiwuww/V‘ED32$<£<m5w5£w omd<303<me3<£920meSousa"? 935 :53(303030228038580 3.4 3.4923szwgd‘mzuzflue: aEéEaH.038: 3383. H .omvmoaé. fiwmowoaé‘ 3883. EEaHumoomofi.< w3-< Hévmwofii HdvmonLq fiovvwoai m-o<. H.mmomm-o< fid~ommé< H.~vomm-o< H.Hfiomm-o< dxx Hwoomméd. m-o< H.mHHmm-Q< HNHommé< 383-3 H.HNomm-O< H.mwm~m-o< 329-3 383-9. $0299. 333-3 H3893 fimmommé< . ‘ 1 71 35323535353mem5$<903<3 35353mest38583358383: 353.5353.0300335303531323 3636:5383:3323323235 $535329:533253353? 8636323:2<mwesm58853<3 mgfioeugmbgss895958,...» §<ma<emv£<m53mea3.25835: 26853:e<eammm5303<mbepm 3286303233330:5me mmSmuwwE/Vfibmwog3.4.35 zazmmw<3<m5583563523? :mwomszmwuanmsgfluflD£03035: :memEOE/xfla $53233 £3mmu53<3<£08<m £<m53m3<mb€3m5u5m asmeaenaébuseoe:533% fiafiuéoegfimw:aaaamzuss 39,0353:me360:9:me 3083: EnuSsu—Dm 3/3535: 858530323253593flay—<83: H.mvmwofi-< H.mwowofi-< H.mewOH-< Hémvwoaé. H.Hmmw0H-< ,‘ H.m~mon-< H.m_m§H-< HKHmwOHuQ H.mHmwOH-< H.Hw¢wOH-< H.momw2-< H.m~mw0H-< fimnmmofi< H.HmmwOH-< H.hmvwoa-< H.mmmm0H.< H.nmmmofi-< H.mvmwofi-< 022335353:eafioefizmzeu ©3233m5NEED392<fl<308<flu 039336.35 <u<w0£<uv5£<$O 8::5:25eueazzgmzefib 83:5thaneueaafizmbefiz 05885833283332 mad::5£03.48:£5<2£um~<mw<fiu mm.:<£<m5mw<£<mw<bw<mbe38.135 035£<MUE<£<£USFD3(3meme 0323858355523:32336 9253<£o8<£<3<§§3”2356 335322:?855 653089268:guts: 0353853233055$333362 om:aeonsEmbasuzmba:9&3: 8::2033235526”?=2m5eo efizgss323523: 8:355:53:£8<53<m52<£< éww5$082933<3<3<m6 dwqmwod,‘ H NwaOH < .E.adommmoaé 3383‘ fi.wwaOH-< H.Nm¢wofi-< Hdeoné aEaEagEgéfivwmwoa < H.~momm-o< Hémfimmé< H.~womm-o< 383.2 333-2 383-3 H.~hmwm-0< H.~oomm-o< H.whomm-o< H.~Bmm-o< Humoommé‘ $89.3 H.829? 33$? #8893 383-3 #8234. 283-3 fiwmommé/x 1 72 8535EDfl<m5uszbeumu330m53 umswmbszuzzs:33<£<8380m53 :msmmzms :flszu—OMS 3285 EOE<MUED£D£DSufism as825553359:amaze:3 3686:5323:$5335ng fizfigéfi<5mmm5 m5 gangs? 85auaussfieasfimsz 392323:«53350382»? 3285:2333:asaogmsefis: 3535.53530943095 3033313233 308<33m Eofiumzmh—Duzmnsanflns 85853035”Sumgeueoe:eds $285Emacssmmwsaa308553 mflDmEDMEmEJEJJED30353035: 3&3mmuSuon/xsbmmzs323535833 3:»me :xamgesmzesmmms325353? 85“35536838635BEES: 3536333558me:5£46533: H.mv¢wOH-< .H.mvmw0H-< dmmmwOHé. HMwaOHLo. 35mg: oai H .Dluwofi-< HHmmwoai H.mwaoH-< ofi-< fianvwofié. $383 $383. H.mmmwOH..< H.R¢wOH-< H.mmvw0H-< H.HmvaH-< H.nvww0H-< H.mvmwofi-< 032$Dou<mwumw<fl<w<w0mw3 :wDflDmtx 832863:3525352363230 09533358:859025283: we:£3828ESEEEonao .853036832322:£33555 83353953:56552355 @328:URsauzeufizngea:5. 53325223:£388: @3230=25azassfibmbefim 8::£§S£<~S£E<S£5ng 8:55«25:68225382382 8532355”Zesufimbmzeue,‘ 0325383285533555 efises<b<3<eu5m5 03:m6£<£<E<e<§age/‘55 095so£<£<203<§<3<m5ego 035£3553<NSSU£<5£<EU @3253:3283662982392 5a:33890303623: :33 .H.Nemm8-<. E E:83 o< EaEa H.mmm~m-o< EaEaééEaEaEaEaEa5%EaE5EaEaEa 1 73 umuDwas3033$0m333mb£<33E45 3M53535308:33832353232: 35we:Eumbubaesmfiaoe::2: ambush: 85323855358535235: :mwwmflufiofin ”$3mobmwsuzwazmuwuaoE<£DE<~ mmSmmSmSmSesmmegsuzfiassz 863530986:sasaaeseaefi §<§<Eo£<ubu=«Gama:3&5: 328535 36263:8333353235.535: 3m¥<me<£<euaaassessmzasmzs 853.5803: 3:1kame hD353335u:£<mb£<£<£<m SUE:3<Eombu35363553? UmemmvSEGED wmuwmuhuénzwauvassuwb . $603803 m5 3 ~35th 303m3383 £<m5 328,035 3 m BUEDmmuSuUmSEDNEN 3233535803332: mh<mbamw5£<mw<fl<£<3 5.483333 mw<£U8<8<m HKHmwOHLQ flammmofié. OH-< H.mvowofi-< HNmmwOHLq H.mwmwo.n-< H.mommofi-< HonwOHLc. H.Hmmon-< H.moowofi-< HzfluowOHLq fimhvonLq 3E8: H.mmwwOH-< H.Hmmwofi-< Hkmmwoglx fiuq H.momwOH-< fimmmonLq 092323303:85525332,»? o9:523,32awmszzeaubmzuz 03232852:besuzufimsmbm2 @3233:«2339505335m2 @228£0£<96$3§<3<£um5aw. was3553285653:£363: was:23:2323923636338 omd<e<eueoe§sageing8 035a:so£<£u£<§<w6ubu5m2 magnum:£523326995025 025:5gaugeosgzms«Ems omd<£<3<£<eaessueweambg £<=b omd<3<£uwwofl< @3335 omd: 035 $35 :5 303595 34.”? wmd<€<2035 303qu mzuwowfiwmzwz m5 :5 £5 2 8 D 20308053me mk<£<mw$<wwmb 022 :5 EU 36. :5 3<mw<u5m¥< m5 3< 8U Easmvmzmbmzmz :2me 2328<2<8<£U FREQ EaééEaEaééEaSomme: H.oovwofi..< HdmmwOHé. Héowwoaé. Hdwowoan HuwnvaHLq dvgwofi< dwwwwOHLfl H.0mmwofi-< OH-< OH-< oaé‘ H.Nmmwofi-< 3839‘ flooommé< H.N:mm-o< 28$? H.HNHmm-O< 388.2 38mm? fidvfimméxx HNmemé< fioma mmé< H.m0Hmm-Q< H.Hwomm-o< H.wHHmm-O< 1 74 353535zmusw<mm9wumw0mw<mw<mw<s 3285 3mu<mUmD :28: 33 368308035328st ufl<mm53<u5203mm5E<£035m5 mflumEDuEEDEOmmE::53<£u:5: 82mg3”5:835:mE<uU£O£<PSa mm»:msswh—szszsmmbswn 3&3m30m585533m533 323230363:m::53<m5 DwONwUDuUUMJwDDw<Mw<3wD m ESE/BEN 303395: 3533308090£658335:234.: §<mm§3oaueuwmepa:Eueoeam $585$:eagmseaaueoeams3 SEE328533395385:5: 828589589835:532?: 3285EDSOMEMMmu—DUHSEUEDPS3 83353383: 30333 3 363963030suumzewfloflwzs03m 35323235swwmzmssbfloisssz $28635eufiommmzsbeaus=5: aaaE . flammmoai H.:mwofi-< H.nwmwofi-< H.mmmon-< EwaoH-< ‘ 3383. aa wmd<swu2335m5m5553<m~<£<£< ©9235 $58355 maze/m5.6£<£<b§£<mbsbe< maze/$35Ewe/SEMGMS30me $353035 @3280mb<ww<fi<£2$<£o€w~b£< 334480803958030953.5 5£<£<Ubfi<mw§ueu 092$: 85353293 3<£§o”2652303239 EEEESEMGESU m5£<m555<3<33 mmd<8<eoe<mb352E033 whom; geogfissae:«2830 :53: 339555832955 omd<fl<£<ww<m5£DUUEUBOE/‘EO @922me£<£o£<§<3<2§3m2 83502532335:8359232 mmd<£<£<es2<£<§ouw<u5£<£u 23536855USEEUEEUUEME 83352::E<£uk<5£<£<a<a< 8:523w£32388863523: donmwOHLq fidwmmofié‘ fiwmmwoflua 3282 3%83.‘ HKmommé< E a5EaEE388: H.mmomm-o< fiwmmmmé/u Ea EE 358.5:eugmseaeagmbeas $58653535395538235: 2685awefibmfizea5:68,; mmzmuqumSEDmmmS 3$<m863380$0833 3.335 umb‘m :5303033m SUSHHE::SuamEaSEmGs? 35320580me3:36.833: 33803 EDEDEDume 35353080353m8<3535ubm53 §<£G£§<£ossm53538205: 3.58235202:35:2?622:m we:98502358255=5323% was35923285853:32352: £0meme333580308332: uflomaaeums3<mmmbeze§Geas fiamuzmbe:sommeomzeueneaz 303084053338829(th3 J£<mfiuflwfl<33:uEGmEEUEOEDm umemmu—ONEEDsuuswmw<mbm¥0w5m5: wflwmmZmS8<mbou303<£u£0£<3 aE.H.mmmm3-< E H.mmmon-< H.nmmon-< H.nwmon-< H.mmmon-< H.Hmvon-< HfihomOHLq aEEEaéaH.HomwOH-< HKvmmOHé. 352<£S<5m5m5m5$< gmzewszmzmzeam2 wasa:£<m2£<e28:Huefififlo 025EEGEEEDSS«255$ mmd<3<8u3033385036.83EUEG wmd<£0mw<£<m5323230308030 mad: fl0mh<£u£<£<w90uhvu5£496 omd<£umw0£<£<32Dw<£<80wu<m5 03280Eofl<£<fluu<w<u5 303305 mmd<3<m5m5u5ESE/HEDEDEDED m32832gamubbbszaumsm2 m9:soe<$<au£2ubw2NEWSEu was£363632355230363230 8::Eu3<£<~Ee920343638 @3255=2a<es203w3<2u£o @325:stave:32.09033205 was805$:358<§<£<=5§m5 @32me£85szEaUEmEE/HEDULD @3233333033859033303035 .H H NnowOH movaH < < H.vmmon.< H.ooowOH-< H.wmmwOH-< H.0mwon.< H.0wmon.< H.wmowOH-< HdmvonLq H.mnomoH-< 3882. -< n-< H.MNHmm H H.mmmNm-D< HémHmméxx H.wmm.~m-o< H wHomm H.mmH D< o< NNHmm-o< H.00Hmm-o< H.wNHmm-Q< m-o< mm-o<. 5%EaEaE55ééEaHJKmNmA—d. H.mthm-o< 1 77 Wowmxam 38038298an 62820:; o~o=¢JN rwdv $028203: ,3 .ncww: 82820:: USA—mo 858953 a 3:865 528a $9381.22 ohém nm.mn mwfiémfi Hm~-mo~ wwwéom mmméfim hm-mm vaNmH mmm-mn~ vom-~vm whém oofiémdn vaNNH Stanza—Bo dogwiconv .mmmnfiw: mo 3230 8533 .620 3230 E 58:8 mo: a. Eocemmnam you 3:335. .53 cum. . , ow U<<OD®DD<<DDUO<OO<<0<<< <<<<D<<U<<OO<0<DU<<D<<< <<U®<D<U<<DUDOUD<U<JDDD <UUUDGD<UU<O<<DDDUDG<<< <<ODDDDDO<ODDGD<D<<<UD< DDDOODU<UDDD<ODDDUDDUDD <<ODODD<<DDUO<OO<<0<<<< 0<<DUDOUD<U<DDDDD DG<ODDOD<D<<<UD<ODU<O<< UUDDG<UDUDDUDDO<DOD<D<D <ODODD<<DDUG<GO<<O<<<<< UDOUD<U<DDDDD<<<<DUO$DD gimme >2: D<0<OODUDuOODDDD<ODDUD< :2: wE mar—U72 E83 8:335. mzom=o ~.B$S.< ammwoz $883. $3on 3382 Nswmwog ~.momw2-< NSmon $583, ~23on Nfimmo: SS8: qumwog D D .8 2an 80:25am E 3%: $5.68 33.38 :65 E D<OD 3.95 805378 82 35m a. $62833. Awucfimwnnm 353.5 2: 83 mm DDDUDDUUDUO<<DD<<U<UD DDD<DDO<DUDUUDDODD<DD D<OU<G<DD®D<DUO DDU<O<<<DDUD$OD<u<OO D<ODDD<D<U<<UDU<<<<<U <<0<<0<<<UD<<<030<B< DDDDUDDUUDUG<<33<<0<U <<<<<DGD<OU<0<DDGD<DU DD<D<U<<UD <D<D<U<DU<<0<<O<ODU<< DDDDDUDDUUDUO<<DD<<2 22633685680 <<8o<333<<<<<302<8 can 2: wmcum 8:0m 2m _ wEmzou=o HNwaoHé. H.m~wwOH-< fimmwwoaé. aéx fimvwwofiéx EEEEEH 805.6% . 252 H.hmwNm-n—< fiwmmwm H H mmmmm ovomm HAVQNm 82? .8330 o< o< o< o< Bank meéHN mmNéNN mom-mvm NOHévH H mMNKHN 83R %5é%aé%HaEg5 <UU<0<<DDDUDO<<<0<0OD<D 0<ODDGD<D<<<UD<GDU<0<<< U330<UDUDDUDDO<DOD<D<DD w<OODUDUwODDDD<ODDUD<<< DGUD<U<DDDDD<<<<DUOGDD< UU<0<<DDDUDO<<<U<OGD<DD <ODDOD<D<<<UD<muDU<O<<<< 3858383383333: 833583833333 0333383353335 830336883853: 38883335380333 03833363023333 38333833233383: 935838333333 5638383 3.303358302530332 8883235358333 33033353333333 N.mmmmoH-< N.mhmon-< N.HmmwOH-< N.mNmmoH-< N.mvmon-< NHomwOHLq NflhmmoHéx ~.mmmmofi-< N.mHmwOH-< N.HmmmoH-< NKvmoni n-< NdnmonLq N.mmmmoH-< NKHmmoHé N.mmmmoH-< NdvmmoHi N.momon-< N.memoH-< <D<UUDGDDDU<0<<<DDU36 DDDUDG<UD<ODDD<D<U<<U <<D<D<U<DU<<G<<O<ODU< DDD<O<<UD<<<<UUO<O<UU D<<UUO<DDDD<<<<<DGD<0 <<D<UUDODDDnv<O<<<DDUD O<UD<ODDD<D<U<< <<<D<D<U<DU<<O<<0<®DU D<DDDDDUDDUUDUO<<JD<< <DDDD<DUODDD<0<<UD<<< (DDUDODD<U<OOGDDG<UDD DD<D<U<OD<<DD<<<U0000 DDDDDUDO<UD<ODDD<D<U< <<U<DU<<<D<D<U<DU<<O< 3<3333832880<<33 303328388233 333383380285: 3333303333388 UDO<UD<DD DD<D<U H3303 H.Hmwon-< H.mmwon-< H wmmmoH-< H.mmwwOH-< H.3NwwOH-< H.waw0H-< H.oowon-< H.HNwon-< H.memOH-< é H.mvwon-< H.mmmwOH-< H.Hmwon-< Smwwoz H3823 3383 H.Nmo~m-o< H.mmmmm H H H H H H vmon H.0mwmm-o< HfimmNmé/u meomm H.mmm~m-o< ommNm Hommm m m voomm n_< o< o< o< o< o< Q< o< mmmmmé< mommmé< E55%EEE hnmfimm 3? $33 33.8 :3: $8-va 23mm .84; $38 838 33mm 8.32 2.23 238 ”$63 832 23mm “$-30 83mm DO<DOD<D<DDDO<DODDU<GDD w<<<<<3<<u<<00<0<au<<3< D<<<UO<D<U<<DUDOUD<U<DD <<ODU<<UUUDOD<UU<O<<DD .0033ng 0304233 D<UDOD<D<<<03 D DD <<UD<030<0<<<<<D<UD<O<D 0<DOD<D<DDDO<DODDU<ODDD <<<<<D<<U<<00<0<DU<<D<< <<<U0<D<U<<DUDOUD<U<DDD <<UUUDOD<U0<0<<DDDUDO<< D<D<<<ODDDDDO<ODD <03<GDU<O<<<<<D<UD<O<D< <303<3<3330<30330<03333 D.O<ODDO<ODDDDO<< <<0<DO<<ODDODDDDDU<UDDD DUDDUDUODDO<DDG<DDO<<DD DDDDDODDUD<DD<DUOD¢ODDD ODUGD<DU<QDDD<DDDDUDDD< DQD<D<DDDG<DODDU<GDDDDD . ‘ NflmmmOHi mammov,‘ N..mmmw8-< Nammwo: Somme: Nmmmmoz “.mmmmoai NAwaoaé. NSmmwOHLQ ~.mmmon-< ~.mmmwOH-< ~.mmmwofi-< 3882 N538: 3382 ~.m3w8-< ~.mmvon-< Néwvwofii ~.movwofi-< <<UDG<<U<DU<<<D<D<U<D 33333833032332: <<202<8<o<303<8o3 <<<383ooa<u<owwzao<u <2<u<w3<<3<<< <82<o3<3383o<us<o <<<uso<<u<8<<§<a<u< 38383333: 25352338380: 333238383668 <58<<<<<u==2<3<u<op 3835332338853 o<<u<8<<§<3<u 38<<<<u8<<u8<<o33 <<<o3w<<<<<u<<u28<8 <<38<<8<<3<<uo<o<< <<<u3<uo<a<<a<w<<u<<< <<<3<<<uao<a<uo 32 H.N©wwOH-< EaEaEaEaEaE5 H.¢Nmmofi-< 2383. 3%éé 3383. 3883 H.Nnm~m-o< E5 E5EaEa.EEEaéééé wwvéov EgE% Ewmvénw mmo H3» How ova m3 Do Hm©-m~m Nmménm mav Hhvévv an? mam mmwékm mavémm HNv 6.5330533053223252: <o<=o<<o=ao3338<u33< 23233252 <=uon<o33828<< DUGD<DU<ODDD<DD 0365453 DDUD DO<DGDDU<ODD DD<D DD D< <UDDO<ODDO<ODDDDO<<UDDD 23253303385335 DDUDUODDD<DD®<DDO<<DD<< <DDDGODDODUOD<DU<GDDD<D D<DU<ODDD<DDDDUDDD<DUDD ODDU<ODDDDD<UDDUDUU<DDD DDO<<UDDDU00<O®<DUDDUDD 330333330<UDDD<3<V DUODDO<DDO<DDD<<DD<<ODD DDDOODDODUOD<DU<ODDD<DD DDUDDOOGDODUDDD<<<0<w<D DDUDD<D<U<ODO<<UDDO<ODD 0<<UDDDUOO<OO<DUDDUDDDD 3383‘ EE% mmwvmofi< N.movaH-< ~.HN¢on-< Nflmvwofiéx 3383 Ndowmofii ofié. ~.hovw.oH-< N.vawo.n-< ~.mmvaH-< N.mmvwofi-< NHDvaHLQ Nflwwwoaé. Naovwofi,‘ N.vawOH.< <<Ossu<<<<uau<<03u<<o ,:<<<wao<<<<<u<<u38<: 3<<38<<8<<8<<uo<o< anuu<m<<<u3<uo<3<<2<o <3<<<o<<<<3<<<uso<3<u UDO<<U<DU<<<D<D <<<ODDU<<<<UDU<<UDU<< <D<<<030<<<<<U<<UD (DUDUDD DU< DD<<DDu<<Du<<DU<<UO<O <D<<<UDO<D<UD<U<<UU<< <<O<D<<<O<<<<D<<<UD®< <0<<03<<<<<UDO< <<o<<O<DUUDUUO<<<ODDU J<D<<<ODO<<<<<U<<UDDU <<UDD<<DDU<<Du<<DU<<U <<D<<<UDO<D<UO<U<<UU<. D<<<G<U<UUU<<G <<UDU<<ODDU<UDOD<D<<0 <<<<o<<w<DUUDUUO<<<GD dmnwmoaé. H.HmwwOH-< Hdwwwofiéx Hfimmmofi-< flmomwofiéx fimowwofiéx EEaaE HKowwOHé. EaE HKomoné. OH-< fid‘ H.HmmNm-o<. H.Nmomm-o< H.mm©~m-o< Hémwmméxx fimmomméd. H.mmon-D< Hfimmwmé,‘ fiwmmmméx‘ H.mmmNm-D< floonmméxx Héonmmé< H.Nonwm-o< H.mo\.mm-o< H.v0h~m-o< #8509 fidonwmém‘ 323-3 H.moRm-o< H.822: thémv Nomémv va-mfio E%EEé%%%Eéégg%gé /340330: D<D<< UODDO<DDO<DDG<<DD<<ODDD DDOODDwDuOD<DU<ODDD<DDD Bazoooaoau33<<<o<o§< ao<<uaao<oanw<w3 <<u38oo<wo<8383333 03335828383233: ao<sao<<3<<033 308308233<<<o<o§<o<< 32032538533303: 82038383323355 3338388032333: 23o<330<33w<<32<<o323< 33030385333333: 33332833323383 55325333028533 3553530333853: <3<<<8388033o<33o<32 Néwvwofii NKmeoHi N.thwofi.< éaE5a28383838533333:.E éa5EEEéEEa DD<D<<<ODG<<<<<U<<UD D <<<UDD<<DDU<<DU<<DU<< <<<D<<<UDG<D<UO<U<<UU 3<88332<<<o<u<8u<< <<uau<<u8<<033<uaoa <<<<<o<<o<8u8uo<<<u 53253355332? <<<<uzz<<38<<8<<8< <<<§<<<u=o<3<uu<u<<u Doua<ouau33<<<u<u<8 <<<u8<68<<038<uao <<<<w<<w<388 <8<<8<<uo<o<<o§3< 3<<<<uas<<38<<8<<3 <<o<<<<2<<<unu<3<uw<u 3<u<<<<3w2<<<w<<2<<<o 5582585385: <<<<u<<u28<33 <<8<<8<<uo<o<<o<aaa Héwwwofibq H.waon-< floomwofié. 3883‘ EE #3336. dmmmwov,‘ a5a 3383 aéd~omwov< 3383‘ 3383. 338: Hémmovl‘ Hdfihmméxx H.Hfih~m-0< H.NHth-Q< 333-9. 3:3 H.323 3 3 323.3 323-3 H.w:~m-o< 323.2 333-2 333-3 333-9 333-2 333-3 333-3 333.2 :33.2 333.2 wnmémm 33% 33$ 83mm mmmfim 0838 $0308 $2-33 mmmgmm 938 22.88 $3-58 32-8: 83mm 33% Q38 $3-23 08302 mNHH-m0HH UDDDDDODDUD<DD<DUOD<ODD DODUOD<Du<QDDD<DDDDUDDD <GDUDOGGDUODDO<O<ODDU<< 20353202: <833ua<w833330<<03< 8388382232833 soanoznaop<<2<a<< DDOD<<D<D<<UDD<D<<O<<<< ODUDGGODUODDG<0<ODDU<<< D<UUD<GDODDDDG<<ODD<UDD UDUDDUD<D<DO<QGD<DU<UD D DDUDODDODDDOD<<3<D<<UDD <<UUUDDD<ODOUDDD®ODDO<D Baoooauonaguéssuéfi 38385235033281: 53582333353633: 38503863803305 wasooa<<3<a<53< 58335383383333 N.mw<mofi-< Wmnvwogué N.mmvmoa-< ,‘ aaa Ndhmon < N.hmwwOH-< N.mwaoH-< ~.mvmwofi-< N.HomwOH-< N.hhmmo.fi.< Ea35aE <<UD<U0<D<<D<0<<U<<<< <<3<<<030<3<00< 330<<UDUDU<<UO<UUU<0< <623<<33<u<oos<82o< 3<<uaau<<<<u<u3<oon<w <3<88<3<2<o<<o<w<8 33<3<33<u<<<u<<u<w<<< DDDDUDD<D<<ODD<D<DD<U DDDO<<UDUDU<<Uo<UUU<O <<OD<<UDDu<<<<u<UD<OO <<ODO<D<UUDU<D<D<O<<O <<ODD<D<DD<U<<<U<<U<0 <30<<UU<<<OU<UD<<<000 3333058825382 58335353550 <<<ua<<u=8<<<<u<us<o 503635322886. 3<<w23<3<3<u<<<u<<u< 3<B<<8<<<8<u3<<<oo .H.mmwwoa-< H.momwoa-< H.wmmmOH-< Ea Himmofi,‘ éé55 H.wmmwOH.< OH-< fimxmwofié. H.mwmmofi-< H.Hmmwo.n-< H.mmmmOH.< $883 fimwnmméxx domn~mé< H H HMthé< 5 HdMNNmé/x HKMNNmAZ dmmhmm o< .ovnmmé/x HHVthé,‘ H.Nvm~m-o< 5%%5 SESAZ 5%EaEa 338 E%5E5E%%3é5%%émmoHéHoH mmoaénoa NwHHéwHH VOWNww .uaoowauozow<o<o:8<<<<< 5835335383833 338323: 5382853803303: noaoziisiuaag 82383333: 32o<w<osau<<<<<wn<u<w§ ounos<<3<<opaos<ousn<3 u<uuu8330<3o233<3<8<< 30693853833053 nonsoszao3<§<a<<u32§< 30583568033533 <8<uoa<uunon<§ u:<<::<<w::o:<833<83< 58828<3o333<3<8<<< O<OOD<DU<UDDUODD<0<DD<< QDDUDDDGJ<<J<D<<UDD<D<< 00.DD<UGDD<OOOUUDDDDODDD D<ODUU<DU<OOD<UUDOD<<DD Nana: Ea5E5aaEE5aaEéN.mmmwOH-< fiuq N.mmmmoH-< N.homwOH-< :3::3.o<<u83<<u058 328325333320 5328538038? <<83<<uo<<o3o<3<83u <3<<oas<3<3<u<<<u<<u .<:§u<<8<<<8<u:<<<o 583633233588 <u<<u3<<32<u< 338335232558 3<<82<<uo<<o8§<83 33333335305 3585888368: <3<u<wu3<uuao<awo<u3 2<w<3<<83<u<533<<33 aaaooa<3<<<u<8<<o<wo DD<<DUD<<UO<<ODO<D<UU 3345426: D<D<DD<U<<<U< <<<U<<<<OOUUUD<<UOD<< <<DD<u<OOD<UUDO<DOD<U H.Hmmwo.n-< fidomwofi H hhmwofi < < H.mwmw0H-< H.mmmwOH-< H.HoomOH-< H.o§a-< 3883 H.vmmmofi-< H.Noomofl-< $88fi< Hfiwmmofli H.mmmwofi-< H.moomofi-< H.vomon-< H.mvn~m-o< Hdthméxx fifimnmmdd. H.Nmnmm-o< fimmnmm Hémhwm H H mmnmm omnmm o< o< EdmmRmAZ o< o< Hfionmmé,‘ . E EEaEa EHavwnmmé/fi 1_85 m~m-mom MHOH meH Hmm mHoH MNOH hmOH 32% MOMH NNVH Ham hmoa-mHOH mmoTvNOH mafia moméwm mom ham HmOH mnofi #9: mHNH-vaH HwNH oovfi <meumham3 mmmaéfimfi hwafifioafi DNHfimHH DD<<GDDOD<OUDD<DUD<UUD< <uu<<uuuow<DUDUDDUD<D<D <003<DU<UDDUODD<O<DD<<D ODDDDD<<D<D<<UDD<D<<O<< DSODD<GOGUUDDDDGDDDUD< ODUU<DU<OOD<UUDOD<<DD<< DD<DUD<UUD<GDGDDD UUOO<DUDUDDUD<D<DG<®OD< UDU<<UUDDUDO<UUDDDUDmVDD DDDGD<<D<D<<UDD<D<<O<<< <u033<0wOUUDDDDODDDUD<< <<0<DO<<UUUD<ODODDDUODD DDDODDOO<DDD<UU<DDD<D<D <UD<GOD<OODDGDUD<<GDUDD UDUUODDD<<<DDDDUD DDDUDU<D<U<U<DDDDD<D<U< UGDD<OOOUUDDDDODDDUD<<< <0<D O<<UUUD<ODGDDDUODD N.mwmwofl-< N.mmmon-< ~.mmmwOH-< fi.< N.nmmon-< fié. H-< Névmwofié‘ NfimmwOHLa N.Mhmwoa-< Ndwmwoaéx N.momwoa-< N.Hmowofi-< N.hmmw0H-< N.mmowofi.< N N.Hmmon-< N mmwwofi< hoowoa-< D<003<0<D<<OUD<U<<UDD <<0<O<DUU00$DDO <DD<<DUD<<UG<<030<D<U DDUDD<D<<GDD<D<DD<U<< D<0<<<U<<<<OOUUUD<<UO .DD<<D.D<U<GOD<UUDO<DOD. <<<U<UD<OOD<O<D<<OUD< D<UUDU<D<D<O<<D<O<DUU <<u<o<<<oo3<o<<ownao :38: 532 33<o<<<u<<<<88u3<<u <<uo<<<u<u3<uuu38<8 <D<D<<<DODD<<<DUU<<u< <<O<UDD<0<U<<UUD<UUD< <0<<<<DDD<<<UOO<ODu<< DODUD<<<<<DGDOD<DG<O< DDD<0<<<U<<<<GGUUUD<< <<<uw<<<u<us<0003 DU<D H..Nnmmo.n-< flowmwofié. H.wwmon.< Hdmmwofii Héoomofié. H.m.mmwo._u-< 3883 H < flammmov< 2883‘ H.moom2-< #2823. fizomofi‘ H.283 mofi-< H.mvomOH-< Héoomoaé. HéfiomoT/x H.won~m-o< fimonmméxx H.onn~m-o< HHNNNmé/x H.-th-D< H.m~.n~m-o< EEEEEEEE .H.anmm-o< H.mwh~m-o< H.vwh~m-o< fimwhmméxx vomTNwNH 33-33 -mm: 58.33 33.22 22-8: $2-82 $3.83 £3.33 583:: :22“: H2302 33.32 33.23 $3.92: $3-22 83m: NmNHéNNH vmma-~mmfi DDGDDGO<DDD<UU<DDD<D<DD 0<<<UDD<UDDG<UDUUODD 0: 30453000333523: GDUDU<D<U<U<DD <030<U<UUDUD DO<UDUUOD UUD UDU<D<U<U<DDD DD 0525825838833: :::<8<:3<:<::o3383 <<<uaa<u3o68uo33<<< 3868833552388: 8323565333323: 8838332<<85<<< DDU<O<GDDDD<UUDDUU<<D<D <<UDD<UDDO<UDUUGDDD<<<D 333033093 DD<<<DD D<< DDUUD D<0<U<D 3 DDUUODD <DD< DDDGDDDUD<<<UU<U<<<< UDU<U<UUDUDDDDDDDOG<DDD DDD<UUDDUU<<D<DO<0<D<DD auq Ndmmwoaé‘ N.mmwwo.n-< NAKooné ~.momwofi-< ~.m~mwofi-< ~.Som3-< ~.SowOH-< ~.m$wofi-< 3&8: ~.:,83-< o.fi-< .~.mvmwofi-< ofi-< N.mnowo.n-< N.nmmwofi-< N.MHomoH-< ~.m~omofi-< V<<D<D<<<DGGD<<<DUU<<U DD<<<UOO<ODU<<OD<<GDD <00<<<<DDD<<<UOO<GDU< <<§o=o3<3w<o .<<<<u.o<<<u<u:<owo:8< <<8<<<u<<3<3<<<oooa< 323380533535: <uuo<<<<333<<<uoo<osu <<303u3<<<<<3o303<no< aazoaooaaa<o<<<u<<<<o <<38<<u<<<<o<owpw=o< <3<338<<ow3<<<682u <333<<<uuo<w8<<oa<<o <<uoo<<<<33<<<uou<os 3<<3o3u3<<<<<poaos<so zaaaoaowga<o<<<u<<<< <<<DUU<<U<<<<0<003030 <<D<DUDU<D<DDOO<<$OD< HNNomoHé. HdmomOH-< H.wmomofi-< EaE5éEaéEEEaaHKHOmOHLQ H.m~omoa-< ~m-o< H.nw.\.Nm-o< H.wwmmm-o< H.mwhwm-o< ESSA: H.~mn~m-o< EEE Hfimhmmé/x éa. Hdmhmméx‘. H.oowmm-o< H.HowNm-o< H.~owNm-o< H.mow~m-o< Héowmméx‘ omvaNVH wmvfiwm»; fi 32-3: mm~H-:~H mme-mmmH Hmwfi-m~vd mmETnmvH HONH-m:H 892$ vmma-~.hma NmVHémvH omvfiémvfi NHNHémHH NomHéwNH mmma-mnma . . <UDD<UDDO<UDUUODDD<<<D D w<UDUUODDD<<<DDDDUUGDD< DU<D<U<U<DDDDD<0<U<DD<D nanosnsua§6u<u<<<<o<3 85533330383322 3<838<<2<3o<o<2<33 33530538033423: <uouuoaao<<<338uoaa<3 waszua<<<uu<u<<<<o<ao<< 33328523: <w<3<332<0383 35385380335233: 88033333388333: <u<<<<o<ao<<u83<onoaaa 22352333 <3<ao<w<3<333<033823 ~.m¢owofi-< fiéx N.hhwmofi-< a5EEE 3883 aa5E5Ea <<DD D<<<UOG<ODU<<DD<< D<<UOO<<<<DDD<<<UOG<O <D<<DODUD<<<<<DODGD<D <Bzzaaoaooaaa<w<<<u< 3<<<8u<<u<<<<o<ooaoa <<<2<88<3<3oo<<ous <<<33<<<umo<oa<<wa< <3<<uoo<<<<222<<<uoo< aaguaaaaoauosangé <3<<<382<<<8u<5<<< <<8<<us<<<<3<88<2< <<<<333<<<uou<osu<<oz <<3<<uoo<<<<33<<<uoo <<<u<ua<ooo38<8333 3<3<<<3ooa<<<8u<<u<< <<<8<<ua<<<<3<38<3 H.mmomoH-< HAvomoHé. $383 H.2omofi-< $882 H.mowNm-o< H.00wNm-n_< :83 3. aEaEaEaEaEa a5%EaEaEaEaEa -HmvH mvfl -Nfimfi Damouvmmfi D<UDDO<UDUUOD DD<<<D 333: 0303030<3<U<u<33333<0<< NAmmoni Numomofi-<.. <<<<<DDD<<<UOO<ODU<<O DJUD<<<<<DODOD<DG<O<U fimmomodhé. advomofié‘ H.HNwNm-Q< H.N~w~m-o< “mucusumm $33333. dongBoQ :ozmwifiou ou=o “8:mean RENO 55-33 3:335. we 32 :58 EZEU 53:8 n. 33 “53803535£<33£0£OE<£08<m 82m£<a<mb£<mm£om5“Ems3% 338583m5£<3u30u5mbm5353 $3qu303¥<ubmwfl<€3353084} Swimsa:33$<m3302<3<£<95m 353583860533803530338:: 82E:m52<m58$<9o£<3<£<m mmuummhfi<3<8<m5uzwuué<8<33353 36m£953:flammau3<m5£<3<m 853593838::uzsuzgwszszs $636358<Enum£wmh<£0£<£<m §<§<$oeusbumese:amazesm 8535:3933::33<£<m5£.0m53 So5_3.m<Z~—mu 8: :2: 3:832 mEmzom=o ~.B$S-< ~.m~mwOH.< NdmmmoHLq oH-< Nimwofli ~Nwmmofi< ~.momw2-< ~.vawOH-< Nmnmwofi< N.mwmmo.fi-< wofi-<. N598: N.mvmmo.n-< < E088 _ .3 $32883. 82$:an 37.: 35:33 Awucmfiwnnm 855 .va82 we EUEGEUE/xmbP—DS moocoswow EEO 9:26 . 82 an cum. Sa:US38506223533295 5:583Bumsaaubenmssze: 2353:3092623533886 S22$»sz39653032590 2232265352mm 2&9«Em:eéasgoaoefiz Se:5235:6523:3:;me 2£<E<363<m§<6m53632.8 EDWUNESD w<2<3<8<39259<8030308.1 5a:385505862455352 S£0335£<£<§09<3§ueu S353$$<£<303<qu 8:855“ «.83. v.5 2: 03.0 2m «:52 35w 8.5m EaEaa5EaEaEaéEEafiomwmofixx H.wmwwOH-< dmfiwwgé. H58: H.mmmeH-< 358—2 30533.» H H x255 252 D~o~mé< wvmwméxx 3&3 33$. #3on .3 32? o< 3 3 299—. 1 90 abmuzmzeassusmzflfizmbefi wmzmws 3.535 8833030868,} 35mfiofiuauiauséwflams£333: mfl<m303530303335303595 BUSES/‘35absmmzmmemn—D 85353: £<m 358530mm:EDJSmEmEEUEOE:m m5: <m5£<ww<fl<m 3532839530339435365335: 8.13580mwmzmwwmzmzmsm $585afiseuaefififla«bat: mmwwm£6.338<mbuu303<80£0£<3 358580332:mmEDmUJGJSHE/xm mfl:m=w03<e<£<u3mu—GJUEOmEmZm 853635”3303833530833:3 $53:meeugmmgmzngefi was2632335?82%6285m 5529883639:£82333: 335%.;33293559:333% :WSWMSMSe<£us§ne<3<e<£<s N.mmmwOH-< .NfihmwOHi NAmmwoaé. N.m~mmo.n-< N.mvmmofi-< N.Homm0H-< NNNmNOHéH Wmmmwofizq Ndamwofii ~.Hmmon-< NflvmwofiéH N.mmmwo~.-< ~.mnmon-<. .N.mmmwOH-< NKHmwOHéH ~.mmmmoH-< ~.m¢mon-< ~.momwo.fi-< ofi-< w<3<£u3033EESOmEEDEUEO 5358336053055313286.me “3memem5m5£<w<¥0mw<m$<w5£< Sa:£0355£<§<ub$<8§u 523533:3253233325 :35£8.53:228<3<£<385 3339:352%me 2923%?uzeesfifio£95 Ems:523528886843586. 2:53:m55322<3<53<£< 2358339292663:samba: 532328693£5358<mb£0u5 S=5385355263532332 H<£uese<£<es2325?»? 5:23::553565595532 5£08323:323863:332 ENS.5309:monuGaamZuS 5:5 33535 355333929me 30308052333533 H.mvww0H-< HHmwoné. fimmwwofixx égEaEa H.NmmmoH-< EaéEéEEa H.mvwon-< E H.Howon-< H.~NmmoH-< H.ommmoH-< H.mmmmoH..< H.mvwon-< HemmonLq HNmONm fimmmmm H E Hémmwm 383:2 $89-3 H H H H vmomm H83 88m m8? ~65on :omm o< o< o< Q< 3 2 o< o< o< 3333333£38333E<30338<w53 my?)m2£43:mUmmqumwOmwbuufi/‘EDm fifisfififi33853095cues: 32.659235suefibmfige:3 $035:23:saunasmsazawsss 8385awaueommefismbsfimzs u£<§oe<a<azswenfiaeuewens 33.0.85:£<e<m§<$<eu8<2<m mm2“eugefizm8533322:a $286953:mbummEmSeBSwEm 3”53636323233235epmfiusa 86me§ne§<m§<e§seesaw 85E:m5eaeaugfiesuzmyaeas ma3m3<£ue=$<m§om§535mg §<§<ew£<m5395955335: atmefi8635935“Reagan: 353530358:mash—(EUMSEOED: mflDmquJE £080: 3455 :58: m <3<3530m3m53580:5353 NKmmwOH N.mHmon N wvon < < %EE~.mmmw2-< Emma: EéEEEEéN.movmoH-< -< n-< < E% EEUEQmEuEEUEZmSEDEUED SEDEOEDUWDuUu—QDMS335535 283m;88:35:53:£8635 E£<385$o£<§<3mus30mm h<£<u5 2 S 885036. £0m~<£<3$§<£3 m2 3U3<m5m53355339383£0 2935”2.5£39<E<E<a§< 5a<eae<euab§o30:53:35 22<au3<mbm§<5303253: Seueoefiseufinbmsmbeomfi Eageugefieaa323535 588580323223530512 E£<£uewe§s2%;ngab 596.253592263335Eu 22E:953252835£35 35362368me w<fi<usflum¥<3<u<5EOE/BEN; 3/3280 u<£<mw<u5H<£Uw<5£<£<3<3< H.Nowon H mmwmoH < < H.mmwon-< é5%EaEaEaEaEaEaH.0mwon-< 3883 3882 H.Nmo~m-o< H H H mnemm nummm D< éEéE5% o< éEéE 383-2 .mwm~m..0< dmwwNmé< SS3? 1‘92 $235 Ewfloanwmfiass 3535333230»: :mUmeDEDuwd‘mS3333:5963: $535«SEDHEMEBEMS353% mmbmsswzwsaogss3534‘80353 mmw<m30mw<m5303335 JmSmEGMS 3.08605: mflommSwszEDmgS358605 EOEUmeSuZwS 35m umquEUEuwSEmmamfiufln303$..me EOEDEHVPGmEM 3.4: 35803532: asmeaaosSwza.agapefis3% ax235903:ecumefibawe:=2: 3235303:32338535385: 03535385303823:3 3333535euwmmbmzeuezeaa as$229.53:33535535msm swampssweaeogggea2&5: 3:333 35853530:68352<e§<$<= 3.585a<e§<m§<mbea”EMSa ”536:5eumhommmzeuaaaaeas N.mvaOH-< N.mmvwoa-< Némvwoflé‘ ~Kmvmo¢< N.mwvw0H-< 3883‘ NHNvmoHi ofi-< N.mmvwoa-< Ndmvwgé‘ ~.mmvmo.fi-< N.hovw0H-< ~.m~vw2-< Ndmvwofii N.mmvmofi-< Natwmofié. NfimvaHé. Ndovwoaé fi..<_ Esoaa928,38DUNEUSUENG 5m2egoafissuefis5&2 S«23828823328?? 5SURE/‘83“2<U$<a<£3£0 E:283255522530m5m5 5«Em?euaomzbzsbefism5. 4.8588255,“..2 EEEED35555238502 ue<aes<eue§w3o 232365missufiauefibg 230$:£<£o£s$<e§<usu§ 2£<3o$<9<£9328335m2 EggmzsbeDEERE?ab Sa:e<£oem§<§<5£<§eu. 28:5£3:8225923328 Ea:gab3633263323“? 230.63:32859625925 Fab968(me385030345230 w<fl<£0£<9<3805flung—NEED EaEEVEaEaEaEaE.H.ooRm-o.< éEEaEaEaEa.%.H.ooRm-o<‘ EaH.wowwofi..< fiwnwwofi< H.won~m-o< Hdonmmé,‘ 1 93 363233363:aseusussséefi 3635$3323.35:32.2035: 359959868580353235: 358530333335£30303?» EameuemueuefiseoEossmEmSa 8285aseogmmmsus5353:: 35358:gangsbeumssfimss segs=me<=5£<m5333 393230363:msuiseamhnaaa saomu‘ogeue:augmfimzefizm 3282329353ng36303:: uaowmzfloenus3:53.33:3&2: 456asuaeoeamzezmzs zaamuzeamzsswma:3:30:52? flagging»;555385: 3285353:903:20295333m meummaeomzezsmemueamzmseas ‘§<Eom5m2m63w53535855: 8532335583:30823083: E3382 %é NHvaOHi ~.R%S-< N.m§m2-< E Nivwoz EEE% h33<3<£<m5333332835 22353&5euzzeumzeug Egefifisuua§§w£ue§u 5£3505gassombmbeuwz Zeueuge822638.853: EEEEEGNSUU28536.30 23825332253:358%: Eggs:«235522385592 .2&3:gab302553528 5532553EEEESZS 23559233633:52er 2.53:a<£<e<§<£o£855 2.5£<5£§Q<Ggaze:=2 53335EEEEUNESSEU 252$:efisosmzefiomb 5m6£<£<3o2<§<9<~5£50 Eggsbmzeabzggefis mzeesfis.5385 Ems92$:a<eu§<9<eoeaez Héwwoni admwwofié. H.00mw0H-< 3883 dmomwofi,‘ dkmmofix‘ $38: 3&8: H.HomwOH-< fimomon< donwwofii .H.whmwoa-< Emmmofidx Hémwwofié H.Nommoa-< HdewOH-< HAhmeOHLq -< H.538: . . éxx H.HHth-D< H.NHth-o< 33%.? 3:33 323.? 3:32 HHHNNmA—d. fiwahmméxx fimfinmméa‘ Hthmméxx H.Hmh~m-o< H.Nmnmm-o< fimthmA—xx HéNthéxx H.m~h~m-o< Hthmméd. é/x fiwNNNmé/x ,UmSmmGuS895%»?th3084.33 «8359:£<32mmm53o3<mbeam 3535828583:”230:5=2: 303220835803:£0me3 853530353633583335345 85852:coaousasmfizsmzag 3533»?me 353634.33 mfloman Eummmzfloegzwzz 3<mm=5m5£083 mmzmmuoE/xsbmwu:380:5mam—£3 mmSmEOEDEmuzzmzmS 3.4: 8285335BumsmSEDNSEUeE 3.086%flauzuseumsszge:m 86353:353383232835gm 3635ongeommmfimseiéoa:m mesmeoeugeummms£858:m3: ma:E<338<m5=ma<ubss£85m $5855eggsmaaazefibefi. 3.3m mw<UuDE<3<m £Um53<mmmbu53330:5: sWEEBUeagmmefibemuefisa 0H-< NNHmmofié. N.mmmw2-< ~.$mwod< $883 3883. ~.momw2-< $383 ~.mmmmow.< Nfimmoz N.$mw3-< $383 3883‘ Némmwoaé. N.nmmmofl.< ~.mmmwofi-< N.momwo.m-< N.mwmwofi.< N.nom.wofi-< 953333 mDUmwd‘HOEUuU 53<me3<8<$823 8,35 5030 2323325£6233:MUm2 2885£36£<§33<e§ueu 23<£a23m5822528525 2:230£§u£$<6£<u5~25 2386235355583365 23288553583335352 533:2:£<m§<58<£u£om6 5~253359356303:£85 Se<3<55£ue§u£< 535532386553:356,3: :55euflwwsquSmEEoESS 529.5233502835~23: 5a:m¥o3<3<2<§<aue<3<m6 S32385Eégsfiawfiefib 5323280358233352352 2352“?3.030553%wam2 Ea:25230250520«590.96 H.HomwOH-< H momwofi < H.588: %E%E5é$883 H.3mwofi-< EgE $882 .é 3883 3-< 3883 H.m¢nmm-0< H .omhwméd. SmRmAE H.~mRm-o< ammRmAZ 323-3 H3293 SmRmé< HERE? dmmfimé‘ H.323? #8293 #8293 333-3. dmmnmmé‘ 333-2 H.moRm-o< doonmmé’x 323-3 1 96 a.3m~28:2032322335285m $68288me3835963232: «5&5£3205833329332: ”“5353:e<3<=m535m5£<$<m 35395808835EDMSEBSN mmsmubfl3933038530345533} 3503:35338338095303035: umUmmGEDuhDus33.43230809me 3.5mun—<mbu5383mm5u5 3m5m303<m5m5mmu5 35833:: E<E<Eo£<m SUE:gamut:3:36:55ch mafiuzeoefibgscamsssbuss 3:3865:332323 maumefioeéogmsUS«2985: geaubmge:«22:35.5: 33365339083:35:5«Baum gnome:£03053359333583 mammeowzeueuzmm533335:5: 35qugauze:3868335335:{ .é~.mmmm2-< éé% .Ndomwofié. N.mmmmofi-< ~.HVmwOH-< NKmmon-< Ndhmwofié‘ N.mwmwofi-< ~.mommOH-< H.< NNmmon< N.mmowofi-< fié‘ N.HmmwOH-< ~.Nomm0H-< N.m~82-< &meEons850635283: m¥<£w£95863prso :35«296.323625293:mt 55:58:3<m5§<3<=588< S2o£<5£<£<6658a£36 53:5535299236203330 286295SomSEGNSNEMUJE 5was8232326239202:so 2mtggflomSUEuEeOe3aw 538532322923:3:252. 53<E<£§<£<§385:sz 233<£§§2<Gwoe3e35 232232309322386352 Eggs:«6822536832 Eggs:esefisfiogsbg 53.85 £<£<£2w59353$? 5ea£03<m§53§o595m2 2336NEEUNUSEMGMSabs: Emsm5£339o§<£§be<8 E3883‘ #3802 333.3 E;EaEaEaEaEaEaEaEaEaEaEaEa.EE%EEaEa $235.35 3435 3582?? DumUEUUuOEDJEN £030£0333<£<3535803 mmsmusuzszuzuman3534.36023 mfl<m30mm<ubusmagma:309335: :thmsustmSEDmsmS30333083: $293035953598580meEDS/E :mSmMEuSQUMS338465353580: 38362:883333230533: 3032832336535 maumeueumbaauze:Eweamfimss easmubaaeugaé $2853553085535252: 3635530353323535532: 858233253353235233 3035339235usa<£ue§<£<m paame§§usbssea$33633:: 35325:55832363335a SUSS53535853:2:355: $2855;ezmmeaeaaueoeam ~.mmmwofi-< Nmmmmofié. Nénmmofié‘ N.momwOH-< ~.m~mwoa-< N.vamo.n-< < %éEé~.m$w2-<_ E 3<£<£0mw<m5uuh<flmv3<£035 w<wu6£<£<35 $<803<m58< EEOPG3<E<£$20303<30£0 um$<mw<uw<5530303586. u<£U£<mu<£<£2<Sfl<m5EOE< 5:5fi<mbm~0mwoswufl<m5mw<u5 hSwOmw/umzmu:33<w<flumwwflwsb 233335£<2$<5m5m5~53< 5:5m5Ewe:a<§<e§<3<£o Es:5£§<£E<E£3392»: 23$;awho£<392<8<mbeuemu 2:5a<£56th93028925 289%:£<322<£§om233 5~25.63<£$<22o303<96 5a:=63queasmefibefiz 2£382533332903635 Ems£85£<e<55m68<m63< ENSa:gaugzfieuefism2 Sagomzmzfiaas£3330. fiomomoaéx H.wmomOH-< flavomoai H mfiomofi < H.m~omOH-< fifimomofié‘ H.mmomoH-< . 55éEE.EaEEEa.% Hfiwmmmfiz fiwwhmméxx fimwhmméx‘. HAthmAE H.~thm-O< H.mmhmm-o< Hémhmmfiz 333.3 H8203. H.323: SmRmAZ dmmRmé< 5< H.How~m-o< H.~omNm-o< H.mom,~m-o<_ $83.3 383.3 38%-? 1 98 :wUmmemUBUmeJDED 803083335 3&3$3.033DUE/xmmubmbmzmzmzs .smUmEUmUusfia3336:5968: 35385:Uggggmzefias as32:neoeuaeoefidszgeaa uflDmEDEOEDEOmaas£8235 85853:35813333358035 3m5m35m5303<333<u5m5 33m 853653:323353559332: $53595£<£8§<£<eom53<~ 365 2232303358353353093: 3.585353605833358<m5=53 mmbmfl<fl<u¥<30$808:8030353 m5: EDSDN 85853<3<3<333$035395: 323536.5283:3523535a ma3&353238235353235 OH-< Ndmmwofii whamwofié‘ NHmmmoaé fiéx ~.moowofi-< N.Hoomofi-< .~.=won-< ~.mmowOH-< Ndvomofié. N.moomo.n-< N.moww0H-< N.mHmon-< ~.mmwwofi-< Némwwofié‘ 3883 u<3<m5m583£<w<h<m5m5m5m5 EEUEDEDmSMGSSDEE/“.8602 5£<m5£Um»<£<»$<£0£0m5m5 u<£<3<3030m5w<530$<£0me u<£<35E<E<£9m¥<w59280:: ogesss32356»? 5eaegaomsazmag 2352309:sfifiaubsszg imbuzmbefissa8859232 23%.:2328890305,“..29: Reageomzefis:enmzmbwfi 292835£o£o55£<euenep 5m5£35Ewessébuzmbg nafisfizeueue: b<£<£<=53<£<§09<m58<£o 5532m?£<=G§oe<ego£u H.m¢omo.fi-< HdHomoH H.309: HdNomOH H < < < emomak HNomNméd. fimommmé< H.mommm-o< HdHWNmAE HAHwNmAE Eag5EaEaEaEaééEaEaEaEa 50% HE 3x88 mswfiofioczo infirm <meu E 3893-8...“ gamma—50-32.50 as EEHHHH 95.38 Ecoon mum—mu 995: .

Io...— HHHHHHH mJEUZ< E 880%qu .288 A..._m_mu ”2 run. win: 528 >9 323 c.5225 HHHEHHHHHH55H 29% . mo m<Zm_m c.3225 IHEHHHEHHHH $.33— 85602 .momon D”. 3“. a a 2:5? % x5050 m>._.mC\mmmH o< 22.3322 m>._.mC\mmmHD<. $2522 222322 £2522 Hag HHHHEHgaaaaafia HEsoE. IHHHHEHE HHHHHHHHHH hmmmm o< a Emma mvd H 2.0 EHHa gaEH H.hhm~m-o< 38%-? 322: 333-2 £23 E23 fiaag wmmmm- o< HHHHHHHHHHEEHHHHHHHHHEEHHHHHHHH80H'33.2 HHHHfiHHHHHEEHHHIHHHHHE H.252 Hod so mod No.0 mod no.0 so ood EEHEHHEggEH! EHHBEEHE so mod mod Hod No.0 mod H06 86 No.0 mod 8.0 8.0 so 3 so 3.9 so so so mod H06 so 8d HHHHHaafiH H5H 3.0 § flag-HHHEH I5. . mNd gum- 5 EHEHHEgg EHHHHHHEREggEEHHHEHH mmd 5 E :0 E. E as 5 N2 N: E 3 E H HEHHHEHHEEEHHHHHHH H.m8mm-n_< HEHEHEHE EHEHEE. E HE28$: :52 389.2 Esme $09-2 22$: E 3.0 so so mg so so No.0 E H 50.0 HE. IE 0 Hod HHHH. EHH EHHH 00.0 a aEHHHEHHEHHaaaaaag I:3 aH a:5 HHHHHaa so A.E .30 E #00 HRHHER 8o HHHHHHHHHEHHHEHHHHHHHHHHHEEaHHHHHHHHHHEHH -o< H_mmom-o< 383-2 $3-2 £32 $5-2 game H.392 $03-2 502.2 £292 EEHHE so E HHEHHaaa so $0 mmomm- D< HHHHEHHHEafiaé HaaaaaaaaaéHEIHHHHHHHE HHHH. 00.0 aEHElfi- 8.0 so N o HagEggHaggagEEHflagHalalHHHHHHHana mod H06 8.0 E HO.0 38° so E so so so so so HH HE5HE 9: E. HEHEHE a .H.5EBEE-flag! DEC :3 m3 mg E 30 30 E N3 E E E 80 so omd H . _HHEggHRHEggEH!EHHHHHHHHHHHHRHHHHEgg fivwommé< H.mwomm-o< 383.3 $52 $03-2 $5-2 383.2 $02-2 $02.2. 503-2 H.323 H.332: @292 H.592 H532 HHHE 207' 8o .30 so m8 so so so 3° HHHE 8o a86 so EHHEHHHI .0 BEE-E No.0 H, H UN: .86 HE 80 o ~.o. HHHEHHHHHHHHHHHH. HHHHEHHHaHEHHH.o EEHHE omd mud Rd E :3 :6 2: so so 30 ”2 de 8.0 mod 2.0 mod 8.0 E so E 2: E hNd med NS No.0 .

, H HEHEHHEEH HEREHHH. IEEHIE!HE! H.monm-D< 323.? H.833: .H.m3mm-o< 283-3 gamma? H32 3%: 32-2 2:32 332 :32 H.223 _ HEHHHHE No.0 EEEEEEEEEEflEEEE EEEEEEEEEEEEEEEEEE EEEEEEEEEEEEEEEE Hod so so so so so . EEEEE ad 8H. 0: so so m3 so 8H $0 EEEEEEE EEEE . EE E 8H E E EEEEEEEEEEEEEEEEEEEE mmo E EEEEEEEEEEE H.-Hmm-0< §$2 EH22 EEgame EEEE H - 33.2 3:32 Elana-fl-HHHH HVHmm- HHHHEso$0 Q< afiaaaafifiaaéHag?HRH? aaaaafiaaaaéHHH'HHHHHHE ElalaaaaaaéHEIHHHHHHHE Table 12. Dose response'screen results for S GalNac-conjugated dsRNA sequences A subset of active siRNAs from the single dose screen (refer to data in Table 11) was tested in a dose response experiment by free uptake in PCH cells. A subset of these .5 active siRNAs was also tested in dose response in Hep3B cells by transfection. lcso ("Ml _—-n- ——-_ -—-:_ ——-_ —_-_ ---_ —_-_ ——._ _—-_ -___ ——-_ ——-i_ —m-_ -—._ -_—-z_ ——-i_ 03‘.1 21.92 -_ Table 13. Results of single dose screen using seguences listed in Table 10. monmerEvo.1nM STDEvo.025nM “m - —m--— AD 526591 ' 0.000 0.000 0.033 .

AD 52678.1 0.002 0.034 0.000 AD-52670.1 0.002 0.009 0.003 3 0.003 0.005 0.029 0.007 0.073 0.000 1 0.016 0.003 AD 52686 1 0.003 0.014 0.000 45 1 0.59 0.004 0.030 0.003 0.030 0.008 ‘ 0.007 0.011 » 0.037 0.014 mm 0.019 0.026 mm 0.041 0.004 "mm 0.028 0.016 mm - 0.003 - 0.002 —--m ‘ 0.004 0.040 0.012 mm 0.013 0.016 m 0.043 0.015 mm 0.023 0.029 0.005. 0.007 ---Iiii 0.004 0.027 mm 0.038 0.014 . 0.014 0017 0.013 0.0203 0.018 0.012 0.026 0.007 0.030 0.006 0.021 0.019 0.004 . 0.029 0.0227 [0.012 mm 0.021 0.018 0.018 0.033 0.00 0.028 0.002 0.013 0.028 AD-52651.1 0.000 0.000 0.007 ”52641-1 ““172 0°02 AD-52707.1 0.001 0.018 0.032 AD-52671.1 0.005 0.034 0.025 . _AD-52650.1 m0.007 0.013 0.041 --AD-52642.1 0.12 m- 0.015 0.022 0.004 .AD-52675.1--m 0.001 0.018 0.044 AD-52647.1 0.031» 0.008 0.023 AD-52716.1-m 0.010 0.06 0.013 49.1 0.136 0.020 0.006 AD-52677.1 0.059 0.040 0.007 _ -AD-52697.1 0.86 0.012 0.021 0.015 AD-52715.1--m 0.005 0.009 0.022 - - 691.1 0.93 0.036 0.017 AD-52698 1 0.010 0.028 0.000 AD~52672 1 0.170 0.014 0.019 AD 52712.1“m 0.007 0.036 . _o 004 AD-52668 1-mm 0.029 . 0.046 0.026 AD-52681.1--m 0.003 0.034 ‘ 0.039 AD 52702.1 0.658 0.060 1 0.014 '215 1.08 1.03 1.11 1.10 0.95 0.43 0.56 0.54. 0.58 0.31 0.54 0.51 0.54. 0.57 . 0.45 0.57 19.1 0.26 0.68 0.76 0.55 0.70 0.73 0.77 0.62 0.63 0.83 0.72 0.87 0.001 0.026 0.76 0.81 AD 1 0.85 0.002 0.042 0.013 0.88 0.030 0.067 AD-527391 0.83 ~ 0.002 0.008 0.061 AD—527801 0.70 0.012 0.021 0.059 A0-52798.1 0.13 m 0.97 0.006 0.038 ‘ AD-52776.1 0.14 0.029 0.023 AD-527S3.1 0.15 1.09 0.048 0.005 AD-52778.1 0.16 0.067 0.003 AD—52744.1 0.16 m 0.91 0.000 0.049 AD—52750.1 0.16 1.01 0.060 0.055 AD-52774.1 0.17 0.89 0.010 0.017 AD-52803.1 0.18 0.92 0.026 0.040 AD-52821.1 0.18 0.87 0.005, 0.046 0.055 A0-52781.1 0.18 0.000 0.023 AD-52779.1 0.20 0.024 0.016 ‘ AD-52793.1 0.20 0.88 0.025 . .069 ' ' AD-52799.1 0.20 1.01 _0.018 0.010 AD-52761.1 0.22- 0.92 0.024 0.023 AD‘-52768.1 0.22 m 0.97 0.028 57.1 0.23 - 0.95 0.040 0.042 AD-52806.1 0.24 m 0.87 0,011 0.084 0.055 AD-52771.1 0.25 0.98 0.010 0.018 ' 0.048 AD-52802.1 0.30 -095 1.00 0.010 0.019‘ 0.005 AD-52731.1 0.30 -085 0.75- 0001 0.067 0.022 AD—52813.1 0.30 0.98 0.001 0.109 0.014 AD-52742.1 0.31 -0.95 1.03 0.005 0.028 - 10.056 AD-52766.1 0.35 1.00 0.010 0.024 0.044 AD-52732.1 0.039 73.1 0.43 m 0.92 0.029 0.022 AD-52772.1 0.43 1.02 . 0.006 0.000 0.065 AD-52822.1 0.016 AD-52783.1 0.45 m 0.76 0.009 0.036 0.019 89.1 0.50 0.78 0.010 0.053 0.004 AD~52795.1 0.50 0.000” 0.080 0.054 AD-52801.1 0.54 0.79 0.018 0.038' 0.035 —m-———m —-mm_—— mum“— Table 14. Resultsof a dose response screen using a subset of sequences from Table A subset of active ANGPTL3 siRNAs from Table 10 were tested by transfection in Hep3B cells in dose se screens. mICSO (nM) 0.0036 0.0037 0.0048 AD-52673.1 0.0049 AD-52711.1 0.0050 0.0054 0.0058 0.0058 0.0060 0.0062 ' 0.0064 0.0064 0.0066 AD-52683.1 0.0069 88.1 0.0071 AD-52717 1 0.0072 AD-52699 1 0.0073 0.0086 0.0088 0.0093 0.0102 0.0109 0.0120 0.0133 0.0143 ' 0.0161 0.0163 0.0179 0.0180 0.0195 0.0216 . 0.0242 0.0318 Table 15. IDs of duplex pairs for which both an unconjua’ged and a GalNae- conjugated version were synthesized and tested These duplexes have the same sequence and modification pattern.

Unconjugated duplex ID GaINac conjugated duplex ID AD-52964.1 AD-52965.1 _ AD-52967.1 AD-52969.1 AD—52659 1 AD-52975.1 60 1 AD—52976.1 61.1 AD-52977.1 692.1V AD-53008.1 AD 52720.1 AD 53036 1 AD-53037 1 . AD:52721.1 AD-52722.1 AD—53038.1 723.1 AD—53039.1 AD—52754.1 AD-53082.1 AD-52755.1 AD-52756.1 AD-52757.1 AD—52758.1 AD—52759.1 AD-52_760.1 AD-52761.1 AD—52762.1. _ AD-52763._1 AD-52764.1 AD—52765.1 AD—52766_.1 _ AD-52767.1 AD-52768.1 ‘ AD-52769.1 AD-52770.1 71.1 - AD-52772.1 - AD-52773.1 AD-52774.1 AD-52775.1 AD-52776.1 AD-52777.1 AD-52778.1 AD-52779.1 ~ AD 53107.1 AD-52780.1 AD 53108 1 81.1 82.1 ' AD-52783.1 ’ AD-52784.1 85.1 AD-53113.1 AD-52'786.1 AD-53114 1 AD-52787.1 AD-53115 1 AD-52788.1 - AD—53116 1 AD-52815.1 AD-53143.1 AD—52816.1 AD-53144.1 In Vivo Tests Example 3.

Test articles In vivo experiments were ted using dsRNA sequences of the ion.

The dsRNA sequence used in the experiments was GalNac-conjugated AD—52981 , sense sequence: AchquququfAfocAngchququfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCqufaucAfaAququfusUfsg (SEQ ID NO: 842)). The dsRNA ce used as a negative control was luciferase-conj ugated AD— ' 48399B1 (“Luc”, sense sequence: CfaCqufaCfngquaGquchfqugAfL96 (SEQ ID NO: 1728), antisense sequence: qugAfaGquchchfgCngi‘aAngfgsAfsu (SEQ ID NO: 1729)). Also used as a negative control was GalNal-conjugated AD—1955 containing alternating 2’-methyl and 2’ fluoro modifications.

Experimental procedure The dsRNA sequences were tested in C57BL/6 (WT) and oh/ob mice. WT mice received five daily doses of dsRNAs in PBS, Luc at 20 mg/kg, or ANG at 5 or 20 ‘ mg/kg; and ob/ob mice received five daily doses ofNPLs formulated with Luc at 20 mg/kg or ANG at 20 mg/kg. All test articles were administered by subcutaneous injection according to the procedure shown in Figure 1. cally, five daily doses of the test articles were administered on five consecutive days (day 0, 1, 2, 3 and 4), and blood samples were ted 5, 3 or 1 day prior to administration, as well as on days 0, 1, 2, 3, 4, 7, 9, ll, 15, 18, 21, 25, 30, 37, 45 and 50 post-administration. The collected blood samples were used to measure the expression ofANGPTL3 protein using an ELISA assay. Levels of serum triglycerides (TGs), low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc) and total cholesterol (TC) were also measured using an Olympus er.

Results Shown in Figure 2, Panel A, are levels of murine ANGPTL3 (mANGPTL3, protein measured in WT mice after administration of control or ANG at 5 or 20 mg/kg.

Also shown in Figure 2, Panel B are levels of mANGPTL3 protein measured in ob/ob mige after administration of control or ANG at 20 mg/kg. The data tes that, for - both WT and ob/ob mice, administration of ANG results in decreased levels of .15} mANGPTL3 protein, as compared to controls.

Shown in Figure 3, Panel A, are levels of LDL-c measured in WT mice afier administration of control or ANG at 20 mg/kg. Shown in Figure 3, Panel B are levels of LDL-c ed in ob/ob mice after administration of control or ANG at 20 mg/kg, The data indicates that administration ofANG causes decreased levels of LDL-c, particularly in ob/ob mice, as compared to controls.

Shown in Figure 4, Panel A, are levels of triglycerides measured in WT mice afier administration of control or ANG at 20 mg/kg. Shown in Figure 4, Panel B are levels of cerides ed in ob/ob mice afier administration of control or ANG at mg/kg. The data indicates that administration ofANG causes decreased levels of tryglycerides, particularly, in ob/ob mice, as compared to controls.

Shown in Figure 5, Panel A and B are levels of total cholesterol (TC) ed in WT and ob/ob mice, respectively, after administration of control or ANG at 20 mg/kg.

The data indicates that administration ofANG causes a moderate se in TC leVels in ob/ob mice, but not in WT mice. Similarly, administration ofANG causes a moderate se in HDL-c levels in ob/ob mice, but not iniWT mice, as is shown in the graphs in Figure 6.

Example 4.

Test article The effect of a single ion of dsRNA sequence of the invention on the level ofANGPTL3 protein was . The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AchquququfAfocAngchququfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCqufaucAfaAfiiAfiiGfiisUfsg (SEQ ID NO: 842)). PBS was used as a negative control.

Experimental procedure The dsRNA sequences were tested in Human PCS Transgenic mouse characterized by liver-specific sion of full-length human PCSK9 gene. Human PCS transgenic mice were dosed with the AD-52981 or PBS using a single subcutaneous injection. The mice were divided into four groups, each group consisting of two males and two females. Each group received an injection of PBS or a 5 mg/kg, 20 mg/kg or 60 mg/kg dose of AD-52981. Blood samples were collected at day land day 0 prior to dosing, and at 72 hours post dosing. ANGPTL3 protein levels were measured by ELISA and compared to levels at day] andday 0 prior to dosing.

Results Shown in Figure 7, are levels of murine ANGPTL3 protein (mANGPTL3) measured in Human PCS transgenic mice. The data shown is expressed ve to PBS control and represents an average for 2 males and 2 females in each. group. Error bars ent standard deviation. The data tes that administration of a single injection of AD-52981 reduces the levels ofIANGPTLS protein in the mice in a ependent manner, with the dose of 60 mg/kg decreasing the levels of 3 protein more v than five-fo1d(see Figure 7).

SEQUENCES - .

SEQ ID N02] >gi|41327750|ref|NM_014495.2I Homo sapiens angiopoietin—like 3 (ANGPTL3), mRNA TTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATTGAAAATCAAGATAAAAATGTTCACAATTAAGCTCCT TCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTC CAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGA CATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATT TGAICAGTCTTTTIATGATCTATCGCTGCAAACCAGIGAAAICAAAGAAGAAGAAAAGGAACTGAGAAGAA CTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACICAAAACTTGAA AGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAAT TCAAAATCAACCTGAAACTCCAGAACACCCAGAAGIAACTTCACTTAAAACTTTTGTAGAAAAACAAGATA ATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATIAAACCAACAGCATAGTCAAATA AAAGAAATAGAAAATCAGCTCAGAAGGACIAGTAITCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCC AAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTG CTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCT CAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGG AAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAAITTTGGT TGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGG» AAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCT AGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATC ACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCAIGATGAGTGTGGA GAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATC TTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAG AAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAATTTAAACATTAACCTCATTCCAAGTTAATGT GGTCTAATAATCTGGTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGATTTTTTTTATCTTAAAGTCACT GTCTATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCGTTTACATTTCTCAATCAAAATICT TATAATACTATTTGTTTTAAATTTTGTGATGTGGGAATCAAITTTAGATGGTCACAATCTAGATTATAATC AATAGGTGAACTTATTAAATAACTTTTCTAAATAAAAAATTTAGAGACITTTATTTTAAAAGGCATCATAT GAGCTAATATCACAACTTTCCCAGTTTAAAAAACTAGTACTCTTGTTAAAACTCTAAACTTGACTAAATAC AGAGGACTGGTAATTGTACAGTTCTIAAATGTTGTAGTATTAATTTCAAAACTAAAAATCGTCAGCACAGA GTATGTGTAAAAATCTGTAATACAAATTTTTAAACTGATGCTTCATTITGCTACAAAATAAITTGGAGTAA 40 ATGTTTGATATGATTTAITTATGAAACCTAATGAAGCAGAATTAAATACTGTATTAAAATAAGTTCGCTGT CTTTAAACAAATGGAGATGACTACTAAGTCACATTGACTTTAACATGAGGTATCACTATACCTTATT SEQ ID NO:2‘ >gi|297278846|ref|XM_001086114.2| PREDICTED: Macaca mulatta angiopoietin—like 3 (ANGPTL3), mRNA ATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGTTGCTTGAAATTGAAAATC AGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGACCA AGACAATTCATCAITTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAA TTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCCAAATT AATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCACTGCAAACCAGTGAAAT CAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACIACAAGTCAAAAATGAAGAGGTAAAGAATA TGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATICTACTTCAACAAAAAGTGAAA TATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACATCCAGAAGTAACTTC ACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGIGGAAGAACAATATA AGCAATTAAACCAACAGCACAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTCAAGAA CCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAATGAAAT AAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAATAGAGGTGAACATATAAGTG GCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTAAAACC TGTCTAAGGAGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTCGGGAG GCTTGATGGAGAATTCIGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTACGTTTTAC GAATTGAGTTGGAAGACTGGAAAGACAACAAACATIATATTGAATATTCTTTTTACTTGGGAAATCACGAA ACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATIT GGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTATTCAGGAGGCTGGT ATGATGAGTGIGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAACAAAATCTAAGCCA GAGCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTI GATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAAATTAAACATTA AACTCATTCCAAGITAATGTGGTTTAATAATCTGGTATTAAATQCTTAAGAGAAGGCTTGAGAAATAGATT TTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCATTTAC ATTICTCAATCAAAATICCTACAACACTATTTGTTTTATATTTTGTGATGTGGGAATCAATTTTAGATGGT CGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAAACTTAGAGACTTTA '30 ATTTTAAAAGTCATCATATGAGCTAATATCACAATTTTCCCAGTTTAAAAAACTAGTTTTCTTGTTAAAAC TCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTAATATTAATTTCAAA ACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCIGTAATATAAAITITTAAACIGATGCCTCATTTTG CTACAAAATAATCTGGAGTAAATTTTTGATAGGATTTATTTATGAAACCTAATGAAGCAGGATTAAATACT GTATTAAAATAGGTTCGCTGTCTTTTAAACAAATGGAGATGATGATTACTAAGTCACATTGACTTTAATAT GAGGTATCACTATACCTTA ' SEQ ID N023 \ >gil142388354lreleM_Ol39l3.3l Mus musculus angiopoietin—like 3 l3), mRNA 40 GGAGAAGTTCCAAATTGCTTAAAATTGAATAATTGAGACAAAAAATGCACACAATTAAATTATTC CTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTC AGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTC ATGGACITAAAGATITTGTCCATAAGACTAAGGGACAAATTAACGACAIATTTCAGAAGCTCAACAIATTT GATCAGTCITTTIATGACCTAICACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAAC 45 TACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGA GICTGCTGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATT CTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTTTTGTAGAACAGCAAGACAA CAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAAITAAGTCAACAGCACATGCAGATAA AAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCA AGAGCACCAAGAACTACTCCCCCTCTTCAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGC CGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATIAAACCAAGAAACTCCC AAGGGTTTHATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGC TCACAGGACTICAACGAAACATGGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGATGGAGAATTTTGGTT GGGCCTAGAGAAGATCIATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGA AAGACAGCAAGCACTACGTTGAATACTCCITTCACCTGGGCAGTCACGAAACCAACIACACGCTACAIGTG GCTGAGATTGCIGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCIGATGTTTTCTACATGGAATCA CAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAG AAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTAC TGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAAICATCCAAAATGATGCTCCAGCCCACCACCTAAGA AGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGT AATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGA TCAGATATCACCGAATCAACGTAAACAAAATTTATC 4 SEQ ID NO:4 >gi|68163568|ref|NM_001025065.1I Rattus norvegicus angiopoietin—like 3 l3), mRNA GACGTTCCAAATTGCTTGAAATTGAATAATTGAAACAAAAATGCACACAATIAAGCTGCTCCTTTTIGTTG TTCCTCTAGTAATTTCGTCCAGAGTTGATCCAGACCTTTCGCCATTTGATTCTGTACCGTCAGAGCCAAAA TCAAGATTTGCTATGTTGGATGATGTCAAAATTITAGCCAATGGCCTCCTGCAGCTGGGTCATGGTCTTAA '25‘ TGTCCATAAGACAAAGGGACAAATTAATGACATATTTCAGAAGCTCAACATATTTGATCAGIGTT TITATGACCTAICACTTCAAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAACCACATCTAAA GTTAAAAACGAAGAGGTGAAGAATATGTCACTTGAACTGAACTCAAAGCTTGAAAGTCTACTGGA GGAGAAGATGGCGCTCCAACACAGAGTCAGGGCTTTGGAGGAACAGCTGACCAGCTTGGTTCAGAACCCGC CTGGGGCTCGGGAGCACCCAGAGGTAACGTCACTTAAAAGTTTTGTAGAACAGCAAGATKACAGCATAAGA GAACTCCTCCAGAGTGTGGAAGAACAATATAAACAACTAAGTCAACAGCACATTCAGATAAAAGAAAIAGA AAATCAGCTCAGAAAGACTGGCATTCAAGAACCCACTGAAAATTCTCTTTATTCTAAACCAAGAGCACCAA 'GAACTACTCCCCCTCTTCATCTGAAGGAAGCAAAAAATATAGAACAAGATGATCTGCCTGCTGACTGCTCT GCCATTTATAACAGAGGTGAACATACAAGTGGCGTGTATACTATTAGACCAAGCAGCTCTCAAGTGTTTAA TGTCTACTGIGACACCCAATCAGGCACTCCACGGACATTAATTCAACACCGGAAAGATGGCTCTCAAAACT AAACGTGGGAAAACTACGAAAAGGGTTTTGGGAGGCTTGATGGTAAAGTGATTTCCTTGCATCAC TCACTTATCTGTTGATTTAATAGTATTAGTTGGGTGTGTTGACACAGGCCTGAGACCATAGCGCTTTTGGG CAAGGGGGGAGGAGGAGCAGCAGGTGAATTGAAAGTTCAAGACCAGTCTGGGCCACACATTGATACTCCTT CTCGACATTAAGAATTATAAATTAAGCAGCAATTATAAAATGGGCTGTGGAAATGTAACAATAAGCAAAAG CAGACCCCAGTCTTCATAAAACTGATTGGTAAATATTATCCATGATAGCAACTGCAATGATCTCATTGTAC TTATCACTACTGCATGCCTGCAGTATGCTTGTTGAAACTTAATTCTATAGTTCATGGTTATCATAAGTCTT 40 ATTAAGGAACATAGTATACGCCATTGGCTCTAGTGAGGGGCCATGCTACAAATGAGCTGCAAAGATAGCAG TATAGAGCTCTTTCAGTGATATCCTAAGCACAACGTAACACAGGTGAAATGGGCTGGAGGCACAGTTGTGG TGGAACACGCGGCCAGCAGGACACTGGGACTGATCCCCAGCAGCACAAAGAAAGTGATAGGAACACAGAGC GAGAGTTAGAAGGGACAGGGTCACCGTCAGAGATACGGTGTCTAACTCCTGCAACCCTACCTGTAATTATT CCATATTATAAACATATACTATATAACTGTGGGTCTCTGCATGTTCTAGAATATGAATTCTATTTGATTGT 45 AAAACAAAACTATAAAAATAAGTAAAAAAATAAAAAATAAACAGATACTTAAAATCAAAAAAAAAAAAAAA AAAAAAAAAA ‘ SEQ ID NO:5 Reverse Complement of SEQ ID NO:1 AATAAGGTATAGTGATACCTCATGTTAAAGTCAATGIGACTTAGTAGTCATCTCCATTTGTTTAAAGACAG TATTTTAATACAGTAITTAATTCTGCTICATTAGGTTTCATAAATAAATCATATCAAACATTTAC TCCAAATTATTTTGTAGCAAAATGAAGCATCAGTTTAAAAATTTGTATTACAGATTTTTACACATACTCTG TGCTGACGATTTTTAGTTTTGAAATTAATACTACAACATTTAAGAACTGTACAATTACCAGTCCTCTGTAT TTAGTCAAGTTTAGAGTTTTAACAAGAGTACTAGTTTTTTAAACTGGGAAAGTTGTGATATTAGCTCATAT GATGCCTTTTAAAATAAAAGTCTCTAAATTTTTTATTTAGAAAAGTIATTTAATAAGTTCACCTATTGATT ATAATCTAGATTGTGACCATCTAAAATTGATTCCCACATCACAAAATTTAAAACAAATAGTATTATAAGAA TTTTGATTGAGAAATGTAAACGGTATTCTTTAAGGTTATGTGATTGTATGTTTAATCTTAAATAGACAGTG ACTTTAAGATAAAAAAAATCTATTTCTCAAGCTTTCTCTTAAGGATTTAATACCAGATTATTAGACCACAT GGAATGAGGTTAATGTTTAAATTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTCTGA ATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCAITTTGAGACTTCCAAGATA ATCCTCTTCTCCTCTCTGGCTTAGATTTTGCTCTTGGTTTGTTATATTTACCATTTAGGTTGTTTTCTCCA CACTCATCATGCCACCACCAGCCTCCTGAATAACCCTCTGGACAGTTGAAGTGTCCTTTTGCTTTGTGATC CCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCGCAACTAGAT GTAGCGTATAGTTGGTITCGTGATTTCCCAAGTAAAAAGAATATTCAATAIAAIGTTTGTTGICTTTCCAG TCTTCCAACTCAATTCGTAAAACATAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAACCA AAATTCTCCATCAAGCCTCCCAAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATCCAT CTATTCGATGTTGAATTAATGTCCATGGACTACCTGATATAACATCACAGTAGACATGAAAAACTTGAGAG TTGCTGGGTCTGATGGCATACATGCCACTTGTATGTTCACCICTGTTAIAAATGGTGGTACATTCAGCAGG AATGCCATCATGTTTTACATTTCTTATTTCATTCAACTGAAGAAAGGGAGTAGTTCTTGGTGCTCTTGGCT TGGAAGATAGAGAAATTTCTGTGGGTTCTTGAATACTAGTCCTTCTGAGCTGATTTTCTATTTCTTTTATT TGACTATGCTGTTGGTITAATTGTTTATATTGGTCTTCCACGGTCTGGAGAAGGTCTTTGATGCTATTATC TTGTTTTTCTACAAAAGTTTTAAGTGAAGTTACTTCTGGGTGTTCTGGAGTTTCAGGTTGATTTTGAATTA TTAGTTGCTCTICTAAATATTTCACTTTTTGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCA GAGTTGAGTTCAAGTGACATATTCTTTACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCT TTCCTTTTCTTCTTCTTTGATTTCACTGGTTTGCAGCGATAGATCATAAAAAGACTGATCAAATA TGTTGAGTTTTTGAAATATGTCATTAATTTGGCCCTTCGTCTTATGGACAAAGICTTTAAGACCATGTCCC AACTGAAGGAGGCCATTGGCTAAAATTTTTACATCGICTAACATAGCAAATCTTGATITTGGCTCTGGAGA ‘30 TAGAGAATCAAATGATGAATTGTCTTGATCAATTCTGGAGGAAAIAACTAGAGGAACAATAAAAAGAAGGA GCTTAATTGTGAACATTTTTATCTTGATTTTCAATTTCAAGCAACGTGGAACTGTTTTCTTCTGGAA SEQ ID NO:6 Reverse Complement ofSEQ ID N022 TAAGGTATAGTGATACCTCATATTAAAGTCAATGTGACTTAGTAATCATCATCTCCATTTGTTTAAAAGAC AGCGAACCTATTTTAATACAGTATTTAATCCTGCTTCATTAGGTTTCATAAATAAATCCTATCAAAAATTT -ACTCCAGATTATTTTGTAGCAAAATGAGGCATCAGTTTAAAAATTTATATTACAGATTTTTACACATACTC TGTGCTGACAATTTTTAGTTTTGAAATTAAIAITACAACAAATTTAAGAACTGTATAATIATCAGTCCTCT TTATTIAGTCAAGITTAGAGTTTTAACAAGAAAACTAGTTTTTTAAACTGGGAAAATTGTGATATTAGCTC ATATGATGACTTTTAAAATTAAAGTCICTAAGTTTTTTATTTAGAAAAGTTATTTAGTAAGTTCACCTGTT GATTATAATTTAGATTGCGACCATCTAAAATTGATTCCCACATCACAAAATATAAAACAAATAGTGTTGTA .40 GGAATTTTGATTGAGAAATGTAAATGGTATTCTTIAAGGTTATGIGATTGTATGTTTAATCTTAAATTGAC AGTGACTTTAAGATAAAAAAATCTATTTCTCAAGCCTTCTCTTAAGGATTTAATACCAGATTATTAAACCA CATTAACTTGGAAIGAGTTTAATGTTTAATTTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTC TGAATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCATTTTGAGACTTCCAGG ATAATCCTCTTCTCCGCTCTGGCTTAGATTTTGTTCTTGGTTTGTTATATTIACCATTTAGGTTGTTTTCT 45 CCACACTCATCATGCCACCACCAGCCTCCTGAATAACTCTCTGGACAGCTGAAGTGTCCTTTTGCTTTGTG ATCCCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCTTAACTA CATGTAGCGTATAGTTGGTTTCGTGATTTCCCAAGTAAAAAGAATATTCAATATAATGTTTGTTGTCTTTC CAGTCTTCCAACTCAATTCGTAAAACGTAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAA ‘ CCAGAATTCTCCATCAAGCCTCCCGAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATC 50 CATCTATICTCCTTAGACAGGTTTTACCTGATACAACATCACAGTAGACATGAAAAACTTGAGAGTTGCTG GGTCTGATGGCATACATGCCACTTATATGTTCACCTCTATTGTAAATGGTGGTACAATCAGCAGGAATGCC‘ ATCATGTTTTACATTTCTTATITCATTCAGCTGAAGAAAéGGAGTAGTTCITGGTGCTCTTGGCTTGGAAG ATAGAGAAATTTCTGTGGGTTCTTGAATATTAGTCATTCTGAGCTGATTTTCTATTTCTTTTATTTGACTG TGCTGTTGGTTTAATTGCTTATATTGTTCTTCCACAGTCTGGAGAAGGTCTTTGATGCTATTATCTTGTTT TTCTACAAAACTTTTAAGTGAAGTTACTTCTGGATGITCTGGAGTTTCAGGTTGATTTTGAATTAAGTTAG TTAGTTGCTCTTCTAAATATTTCACTITITGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCAAGTITT IGAGTTGAGTTCAAGTGACATATTCTITACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCTTCTCAG TTCTTCTTCTTTGATITCACTGGTITGCAGTGATAGATCATAAAAAGACTGATCAAATATGTTGA GTTITTGAAATATGTCATTAATTTGGCCCTTAGTCTTATGGACAAAGTCTTTAAGACCATGTCCCAACTGA AGGAGGCCATTGGCTAAAATTTTTACATCGTCTAACATAGCAAAICITGATTTTGGCTCTGGAGATACAGA AICAAATGATGAATTGTCTTGGICAAITCTGGAGGAAATAACTAGAGGAACAATAAAAAGAAGGAGCTTAA TTGTGAACATTTTTATCCTGATTTTCAATTTCAAGCAACGTGGAACTGTGTTCTTCTGGAAGCAGACCTAG ACTTCTTAACTCTATATAT SEQ ID NO:7 Reverse Complement of SEQ ID NO:3 ' CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAATAATIGAGACAAAAAATGCACACAATTAAATTATTC CTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTC AGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTC ATGGACTTAAAGATTTTGTCCATAAGACTAAGGGACAAAITAACGACATATTTCAGAAGCTCAACATATTT GATCAGTCITTTTATGACCTAICACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAAC . TACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGA TGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATT CTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTITTGTAGAACAGCAAGACAA CAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAATTAAGTCAACAGCACATGCAGATAA AAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCA ' AGAGCACCAAGAACTACTCCCCCTCTICAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGC CGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATTAAACCAAGAAACTCCC AAGGGTTTAATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGC TCACAGGACTTCAACGAAACATGGGAAAACTACGAAAAGGGCTITGGGAGGCTCGATGGAGAATTTTGGIT GGGCCTAGAGAAGATCTATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGA AAGACAGCAAGCACTACGTTGAATACTCCTTTCACCTGGGCAGTCACGAAACCAACTACACGCTACATGTG GCTGAGATIGCIGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCTGATGTTTTCTACATGGAATCA CAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAG AAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTAC TGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAATCATCCAAAATGATGCTCCAGCCCACCACCTAAGA AGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGT AATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGA ATCACCGAATCAACGTAAACAAAATITATC SEQ ID NO:8 Reverse Complement of SEQ ID N014 TTTTTTTTTTTTTITTTTTTTTTTTGATTTTAAGTATCTGTTTATTTTTTATTTTTTTACTTATTTTTATA 40 GTTTTGTTTTACAATCAAATAGAATTCATATTCTAGAACATGCAGAGACCCACAGTTATATAGTATATGTT TATAATATGGAATAATTACAGGTAGGGTTGCAGGAGTTAGACACCGTATCTCTGACGGTGACCCTGTCCCT TCTAACTCTCGCTCTGTGTTCCTATCACTTTCTTIGTGCTGCTGGGGATCAGTCCCAGTGTCCTGCTGGCC GCGTGTTCCACCACAACTGTGCCTCCAGCCCATTTCACCTGTGTTACGTTGTGCTTAGGATATCACTGAAA GAGCTCTATACTGCTAICTITGCAGCTCATTTGTAGCATGGCCCCTCACTAGAGCCAATGGCGTATACTAT 45 TAATAAGACTTATGATAACCATGAACTATAGAATTAAGTTTCAACAAGCATACTGCAGGCATGCA GTAGIGATAAGTACAATGAGATCAITGCAGTTGCTATCATGGATAATATTTACCAATCAGTTITATGAAGA CTGGGGTCTGCTTTTGCTTATTGTTACATTTCCACAGCCCATTTTATAATTGCTGCTTAATTTATAATTCT TAATGTCGAGAAGGAGTATCAATGTGTGGCCCAGACTGGTCTTGAACTITCAATTCACCTGCTGCTCCTCC TCCCCCCTTGCCCAAAAGCGCTATGGTCTCAGGCCTGTGTCAACACACCCAACTAATACTATTAAATCAAC AGATAAGTGAGTGATGCAAGGAAATCACTTTACCATCAAGCCTCCCAAAACCCTTTICGTAGTTTTCCCAC GTTTGGTTGAAGTTTTGAGAGCCATCTTTCCGGTGTTGAATTAATGTCCGTGGAGTGCCTGATTGGGTGTC GACATTAAACACTTGAGAGCTGCTTGGTCTAATAGIATACACGCCACTTGTATGTTCACCTCTGT TGGCAGAGCAGTCAGCAGGCAGATCATCTTGTTCTAIATTTTTTGCITCCTTCAGATGAAGAGGG GGAGTAGTTCTTGGTGCTCTTGGTTTAGAATAAAGAGAATTTTCAGTGGGTTCTTGAATGCCAGTCTTTCT GAGCTGATTTTCTATTICTTTTATCTGAATGTGCTGTTGACTTAGTTGTTTATATTGfTCTTCCACACTCT GGAGGAGTTCTCTTATGCTGTTATCTTGCTGTTCTACAAAACTTTTAAGTGACGTTACCTCTGGGTGCTCC CGAGCCCCAGGCGGGTTCTGAACCAAGCTGGTCAGCTGTTCCTCCAAAGCCCTGACTCTGTGTTGGAGCGC CATCTICTCCTCCAGTAGACTTTCAAGCTTTGAGTTCAGTTCAAGTGACATATTCTTCACCTCTTCGTTTT TAACTTGTAGTTTAGATGTGGTTCTTCTTAGCTCCTTTTCCTCTTCTTTGATTTCATTGGTTTGAAGTGAT TAAAAACACTGATCAAATATGTTGAGCTTCTGAAATATGTCATTAATTTGTCCCTTTGTCTTATG GACAAAATCTTTAAGACCATGACCCAGCTGCAGGAGGCCATTGGCTAAAATTTTGACATCATCCAACATAG CAAATCTTGATTTTGGCTCTGACGGTACAGAATCAAATGGCGAAAGGTCTGGATCAACTCTGGACGAAATT ACTAGAGGAACAACAAAAAGGAGCAGCTTAATTGTGTGCATTTTTGTTTCAATTATTCAATTTCAAGCAAT TTGGAACGTC SEQ ID N019 ~ Macaca fascicularis angiopoietin—like 3 (Angptl3), 'mRNA GGGTAGTATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGCTGCTTGAAATTGA AAATCAGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTICCTCTAGTTATTTCCTCCAGAATT GACCAAGACAAITCATCATTTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGT AAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCC AAATTAATGACATATTTCAAAAACTCAACATATTIGATCAGTCTITTTATGATCTATCACTGCAAACCAGT GAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAA GAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTICAACAAAAAG TGAAATATTTAGAAGAGCAACTAACTAACITAATTCAAAATCAACCTGCAACTCCAGAACATCCAGAAGTA ACTTCACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGTGGAAGAACA ATATAAGCAATTAAACCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTC ‘ AAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAAT GAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAAIAGAGGTGAACATAT AAGTGGCACGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTA GTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAA TTCGGGAGGCTTGATGGAGAATTCTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATC '35 TAATTACGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACT TGGGAAATCACGAAACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCG 'GAAAACAAAGATTTGGTGTTTTCTACTTGCGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTA TTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAA CAAAATCTAAGCCAGAGCGGAGAAGAGGATTATCCTGCAAGTCTCAAAATGGAAGGTTATACTCTATAAAA 40 TCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAA TAAATTAAACATTAAACTCATTCCAAGTTAATGTGGTTTAATAATCTGGTATTAAATCCITAAGAGAAGGC TTGAGAAATAGATTTTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAA AGAATACCATTTACATTTCTCAATCAAAATTCTTACAACACTATTTGTTTTATATTTTGTGATGTGGGAAT TAGATGGTCGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAA 45. ACTTAGAGACTTTAATTTTAAAAGTCATCATATGAGCTAATGTCACAATTTTCCCAGTTTAAAAAACTAGT TTTCTTGTTAAAACTCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTA ATATTAATTTCAAAACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCTGTAATAIAAATTTTTAAACT GATGCCTCATTTTGCTACAAAATAATCTGGAGTAAATTITTGATAGGATTTATTTATGAAACCTAATGAAG CAGGATTAAATACTGTATTAAAATAGGTTCGCTGTCITTTAAACAAATGGAGATGATGATTACTAAGTCAC 50 ATTGACTTTAATATGAGGTATCACTATACCTTAACATATTTGTTAAAACGTATACTGTAIACATTTTGTGT

Claims (40)

We claim:

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3, wherein said dsRNA comprises a sense strand and an antisense strand, wherein said sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides 5 from the nucleotide sequence of SEQ ID NO: l and said antisense strand comprises at least 15 uous nucleotides differing by no more than3 nucleotides from the nucleotide sequence of SEQ ID NO: 5.

2. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ANGPTL3, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand 10 comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10.

3. The dsRNA of claim 2 , wherein the sense and antisense strands comprise sequences selected from the group consisting of AD-52981.1, AD-53063.1, AD- 53001.1, AD-53015.1, 15 AD-52986.1, AD-52953.1, AD-53024.1, AD-53033.1, AD-53030.1, AD-53080.1, AD-53073.1, AD-53132.1, AD-52983.1, AD-52954.1, AD-52961.1, AD-52994.1, AD-52970.1, AD-53075.1, AD-53147.1, AD-53077.1 of Tables 7 and 8.

4. The dsRNA of any one of claims 1 to 3, wherein said dsRNA comprises at least one modified nucleotide. 20

5. The dsRNA of claim 4, wherein at least one of said modified nucleotides is selected from the group consisting of a 2'methyl ed nucleotide, a nucleotide comprising a 5'- phosphorothioate group, and a al nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

6. The dsRNA of claim 4, wherein said modified nucleotide is selected from the group 25 consisting of a 2'-deoxy-2'-fluoro modified tide, a xy-modified tide, a locked nucleotide, an abasic tide, a 2'-amino-modified tide, a 2'-alkyl-modified nucleotide, a morpholino tide, a phosphoramidate, and a non- natural base comprising nucleotide.

7. The dsRNA of any one of claims 2 to 6, wherein the region of complementarity is at least 17 nucleotides in .

8. The dsRNA of any one of claims 2 to 6, wherein the region of complementarity is between 19 and 21 nucleotides in . 5

9. The dsRNA of claim 8, wherein the region of complementarity is 19 nucleotides in length.

10. The dsRNA of any one of claims 1 to 9, wherein each strand is no more than 30 nucleotides in length.

11. The dsRNA of any one of claims 1 to 10, wherein at least one strand comprises a 3' 10 overhang of at least 1 nucleotide.

12. The dsRNA of any one of claims 1 to 10, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

13. The dsRNA of any one of claims 1 to 12, further comprising a ligand.

14. The dsRNA of claim 13, wherein the ligand is ated to the 3' end of the sense 15 strand of the dsRNA.

15. The dsRNA of claim 13 or claim 14, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

16. The dsRNA of claim 15, wherein the ligand is

17. The dsRNA of any one of claims 2 to 16, wherein the region of complementarity consists of one of the antisense sequences of Tables 2, 3, 7, 8, 9 and 10.

18. The dsRNA of any one of claims 1, 2 or 13-16, wherein the dsRNA comprises a sense strand consisting of a sense strand sequence selected from the sequence of Tables 2 , 3 , 9 and 5 10, and an antisense strand consisting of an antisense sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10.

19. An ed and/or non-human cell containing the dsRNA of any one of claims 1 to

20. A vector encoding at least one strand of a dsRNA, n said dsRNA comprises a 10 region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein said dsRNA is 30 base pairs or less in length, and wherein said dsRNA targets said mRNA for cleavage.

21. The vector of claim 20, wherein the region of complementarity is at least 15 nucleotides in . 15

22. The vector of claim 20, n the region of complementarity is 19 to 21 tides in length.

23. An isolated and/or non-human cell comprising the vector of claim 20.

24. A pharmaceutical composition for inhibiting sion of an ANGPTL3gene comprising the dsRNA of any one of claims 1 to 18 or the vector of any one of claims 20 to 22. 20

25. The pharmaceutical composition of claim 24, further comprising a lipid formulation.

26. The pharmaceutical composition of claim 25, wherein the lipid formulation comprises a SNALP, or XTC.

27. The pharmaceutical composition of claim 25, wherein the lipid formulation comprises a MC3.

28. A method of inhibiting ANGPTL3 expression in an isolated and/or non-human cell, the method comprising: a) contacting the cell with the dsRNA of any one of claims 1 to 18 or the vector of any one of claims 20 to 22; and 5 b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA ript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.

29. The method of claim 28, wherein said cell is a non-human cell within a n o n - h u m a n subject. 10

30. The method of claim 28, wherein the isolated cell is an ed human cell.

31. The method of claim 30, wherein the isolated human cell is derived from a human subject suffering from a disorder of lipid metabolism.

32. The method of claim 31, wherein the disorder of lipid metabolism is hyperlipidemia or hypertriglyceridemia. 15

33. The method of any one of claims 28 to 32, wherein the ANGPTL3 expression is inhibited by at least about 30%.

34. Use of the dsRNA of any one of claims 1 to 18 or the vector of any one of claims 20 to 22 in the manufacture of a medicament for the treatment of a subject having a disorder that would benefit from ion in ANGPTL3 expression. 20

35. The use of claim 34, wherein the disorder is a disorder of lipid metabolism.

36. The use of claim 35, wherein the disorder of lipid lism is hyperlipidemia or riglyceridemia.

37. The use of any one of claims 34 to 36, wherein the dsRNA causes a decrease in one or more serum lipid and/or a decrease in ANGPTL3 protein accumulation in the subject. 25

38. The use of any one of claims 34 to 37, n the medicament is manufactured to provide the dsRNA for administration at a dose of about 0.01 mg/kg to about 10 mg/kg or about 5 mg/kg to about 50 mg/kg.

39. Use of the dsRNA of any one of claims 1 to 18 or the vector of any one of claims 20 to 22 in the manufacture of a medicament for inhibiting the expression of ANGPTL3 in a subject. 5

40. The use of claim 39, wherein the medicament is ctured to provide the dsRNA for administration at a dose of about 0.01 mg/kg to about 10 mg/kg or about 5 mg/kg to about 50 mg/kg. Pre-bieed (Ci—S, -3, 4) Bleed (d7, 9, H,15,18,21,25,30,37,45,50) illllli l i ii 5 daiiy doses (mg/kg) and Bieed (d0, d1, d2, d3; d4)