PCSK9 iRNA COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

To provide effective treatments for PCSK9-associated diseases, such as a hyperlipidemia, e.g., hypercholesterolemia.SOLUTION: The invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the PCSK9 gene, and methods of using such RNAi agents to inhibit expression of PCSK9 and methods of treating subjects having a lipid disorder, such as a hyperlipidemia.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 61 / 733,518, filed December 5, 2012; U.S. Provisional Patent Application No. 61 / 793,530, filed March 15, 2013; U.S. Provisional Patent Application No. 61 / 886,916, filed October 4, 2013; and U.S. Provisional Patent Application No. 61 / 892,188, filed October 17, 2013. This application is also related to U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011. The entire contents of each of the above provisional patent applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on October 29, 2013, is titled 121301-00420_SL.txt and is 433,512 bytes in size. [Background technology]

[0003] The proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family. The other eight mammalian subtilisin proteases, PCSK1-PCSK8 (also called PC1 / 3, PC2, furin, PC4, PC5 / 6, PACE4, PC7, and S1P / SKI-1), are proprotein convertases that process various proteins in the secretory pathway and play roles in diverse biological processes (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5).

[0004] PCSK9 has been proposed to play a role in cholesterol metabolism. PCSK9 mRNA expression, like cholesterol biosynthetic enzymes and the low-density lipoprotein receptor (LDLR), is downregulated by dietary cholesterol feeding in mice (Non-Patent Document 6), upregulated by statins in HepG2 cells (Non-Patent Document 7), and upregulated in sterol regulatory element-binding protein (SREBP) transgenic mice (Non-Patent Document 8). Furthermore, PCSK9 missense mutations have been found to be associated with a form of autosomal dominant hypercholesterolemia (Hchola3) (Non-Patent Document 9, Non-Patent Document 10, Non-Patent Document 11). Because single nucleotide polymorphisms (SNPs) have been associated with cholesterol levels in the Japanese population, PCSK9 may also play a role in determining LDL cholesterol levels in the general population (Non-Patent Document 12).

[0005] Autosomal dominant hypercholesterolemia (ADH) is a monogenic disease in which patients exhibit elevated total cholesterol and LDL cholesterol levels, tendon xanthomas, and early atherosclerosis (Non-Patent Document 13). The etiology of ADH and the recessive form of autosomal recessive hypercholesterolemia (ARH) (Non-Patent Document 14) is due to defective LDL uptake by the liver. ADH can be caused by LDLR mutations, which prevent LDL uptake, or by mutations in apolipoprotein B, a protein on LDL that binds to LDLR. ARH is caused by mutations in the ARH protein, which is required for endocytosis of the LDLR-LDL complex through its interaction with clathrin. Therefore, if PCSK9 mutations are the cause in the Hchola3 family, it is likely that PCSK9 plays a role in receptor-mediated LDL uptake.

[0006] Overexpression studies have pointed to the role of PCSK9 in regulating LDLR levels and, therefore, LDL uptake by the liver (Non-Patent Document 15, Non-Patent Document 16, Non-Patent Document 17). Adenovirus-mediated overexpression of mouse or human PCSK9 in mice for 3 or 4 days results in elevated total cholesterol and LDL cholesterol levels; this effect is not seen in LDLR knockout animals (Non-Patent Document 15, Non-Patent Document 16, Non-Patent Document 17). Furthermore, PCSK9 overexpression results in a significant decrease in hepatic LDLR protein without affecting LDLR mRNA levels, SREBP protein levels, or the nuclear-to-cytoplasmic ratio of SREBP protein.

[0007] Although hypercholesterolemia itself is asymptomatic, long-term elevation of serum cholesterol can lead to atherosclerosis. Over decades, chronically elevated serum cholesterol causes the formation of atheromatous plaques in arteries, which can lead to progressive narrowing or even complete blockage of the affected arteries. Furthermore, smaller plaques can rupture, forming clots and obstructing blood flow, resulting in, for example, myocardial infarction and / or stroke. When the formation of stenosis or blockage is gradual, blood supply to tissues and organs is slowly reduced until organ function is impaired. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Bergeron, F. (2000) J. Mol. Endocrinol. 24, 1-22 [Non-patent document 2] Gensberg, K., (1998) Semin.Cell Dev.Biol.9,11-17 [Non-patent document 3] Seidah, NG (1999) Brain Res. 848, 45-62 [Non-patent document 4] Taylor, NA, (2003) FASEB J.17, 1215-1227

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Non-licensed literature 9

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[0009] Thus, there is a need in the art for effective treatments for PCSK9-associated diseases such as hyperlipidemia, e.g., hypercholesterolemia. [Means for solving the problem]

[0010] As described in more detail below, compositions comprising RNAi agents, e.g., double-stranded iRNA agents, that target PCSK9 are disclosed herein. Methods of using the compositions of the invention to inhibit PCSK9 expression and treat conditions associated with PCSK9 expression, e.g., hypercholesterolemia, are also disclosed.

[0011] Accordingly, in one aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, wherein the double-stranded RNAi agent comprises a sense strand that is complementary to an antisense strand, and the antisense strand comprises a region that is complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; Each n may or may not be present p , n p ',n q , and n q ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; N b The modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand is conjugated to at least one ligand The present invention provides a double-stranded RNAi agent represented by:

[0012] In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1.

[0013] In one embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0014] In one embodiment, the YYY motif is present at or near the cleavage site on the sense strand.

[0015] In one embodiment, the Y'Y'Y' motif is present at positions 11, 12 and 13 of the 5'-end antisense strand.

[0016] In one embodiment, Y' is 2'-O-methyl.

[0017] In one embodiment, formula (III) is formula (IIIa): Sense:5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5'(IIIa) is expressed by

[0018] In another embodiment, formula (III) is formula (IIIb): Sense:5'n p -N a -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIIb) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) is expressed by

[0019] In yet another embodiment, formula (III) is formula (IIIc): Sense:5'n p -N a-XXX-N b -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5'(IIIc) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) is expressed by

[0020] In one embodiment, formula (III) is formula (IIId): Sense:5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIId) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N a and N a ' independently represent an oligonucleotide sequence containing 2 to 10 modified nucleotides) is expressed by

[0021] In one embodiment, the double-stranded region is 15 to 30 nucleotide pairs in length. In another embodiment, the double-stranded region is 17 to 23 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 17 to 25 nucleotide pairs in length. In one embodiment, the double-stranded region is 23 to 27 nucleotide pairs in length. In another embodiment, the double-stranded region is 19 to 21 nucleotide pairs in length. In another embodiment, the double-stranded region is 21 to 23 nucleotide pairs in length. In one embodiment, each strand has 15 to 30 nucleotides.

[0022] In one embodiment, the modification on the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In another embodiment, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0023] In one embodiment, the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. [ka] is.

[0024] In one embodiment, the ligand is attached to the 3' end of the sense strand.

[0025] In one embodiment, the RNAi agent is shown in the following schematic diagram: [ka] wherein X is O or S. In certain embodiments, X is O.

[0026] In one embodiment, the agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0027] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand. In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0028] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. In one embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0029] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand. In one embodiment, the strand is the antisense strand.

[0030] In one embodiment, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

[0031] In one embodiment, the Y nucleotide comprises a 2'-fluoro modification.

[0032] In one embodiment, the Y' nucleotide comprises a 2'-O-methyl modification.

[0033] In one embodiment, p'>0. In another embodiment, p'=2.

[0034] In one embodiment, q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA. In another embodiment, q'=0, p=0, q=0, and the p' overhanging nucleotides are non-complementary to the target mRNA.

[0035] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0036] In one embodiment, at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond.

[0037] In one embodiment, all n p ' is attached to the adjacent nucleotide via a phosphorothioate bond.

[0038] In one embodiment, the RNAi agent is selected from the group of RNAi agents listed in Table 1, Table 2, Table 9, Table 10, Table 12, and FIG.

[0039] In one embodiment, the RNAi agent is selected from the group consisting of AD-53815, AD-56663, AD-56658, AD-56676, AD-56666, AD-57928, and AD-60212.

[0040] In another aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, wherein the double-stranded RNAi agent comprises a sense strand that is complementary to an antisense strand, and the antisense strand comprises a region that is complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; Each n may or may not be present p , n p ',n q , and n q ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand is conjugated to at least one ligand The present invention provides a double-stranded RNAi agent represented by:

[0041] In yet another aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, wherein the double-stranded RNAi agent comprises a sense strand that is complementary to an antisense strand, and the antisense strand comprises a region that is complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i-N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand is conjugated to at least one ligand The present invention provides a double-stranded RNAi agent represented by:

[0042] In a further aspect, the invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, wherein the double-stranded RNAi agent comprises a sense strand that is complementary to an antisense strand, and the antisense strand comprises a region that is complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; The present invention provides a double-stranded RNAi agent represented by:

[0043] In another aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, wherein the double-stranded RNAi agent comprises a sense strand that is complementary to an antisense strand, and the antisense strand comprises a region that is complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-Nb '-(Z'Z'Z') l -N a '-n q '5'(III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modification on Y is different from the modification on N b The modification on ' is different from the modification on Y'; the sense strand comprises at least one phosphorothioate linkage; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; The present invention provides a double-stranded RNAi agent represented by:

[0044] In yet another aspect, the present invention provides an RNAi agent, e.g., a double-stranded RNAi agent, capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, wherein the double-stranded RNAi agent comprises a sense strand that is complementary to an antisense strand, and the antisense strand comprises a region that is complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by formula (III): Sense:5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-n q '5'(IIIa) (In the formula: Each n may or may not be present p , n q , and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; the sense strand comprises at least one phosphorothioate linkage; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; The present invention provides a double-stranded RNAi agent represented by:

[0045] The present invention also provides cells, vectors, host cells, and pharmaceutical compositions comprising the double-stranded RNAi agents of the present invention.

[0046] In one embodiment, the invention provides an RNAi agent selected from the group of RNAi agents listed in Table 1, Table 2, Table 9, Table 10, Table 12, and FIG.

[0047] In certain embodiments, the RNAi agent is administered using a pharmaceutical composition.

[0048] In a preferred embodiment, the RNAi agent is administered in a solution. In some such embodiments, the siRNA is administered in a non-buffered solution. In one embodiment, the siRNA is administered in water. In other embodiments, the siRNA is administered with a buffer such as acetate buffer, citrate buffer, prolamin buffer, carbonate buffer, or phosphate buffer, or any combination thereof. In some embodiments, the buffer is phosphate buffered saline (PBS).

[0049] In one embodiment, the pharmaceutical composition further comprises a lipid formulation. In one embodiment, the lipid formulation comprises LNP or XTC. In another embodiment, the lipid formulation comprises MC3.

[0050] In one aspect, the present invention provides a method for inhibiting PCSK9 expression in a cell, the method comprising contacting the cell with an RNAi agent, e.g., a double-stranded RNAi agent, or a vector of the present invention; and maintaining the cell produced in step (a) for a time sufficient to result in degradation of the mRNA transcript of the PCSK9 gene, thereby inhibiting expression of the PCSK9 gene in the cell.

[0051] In one embodiment, the cell is in a subject.

[0052] In one embodiment, the subject is a human.

[0053] In one embodiment, PCSK9 expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0054] In another aspect, the present invention provides a method for treating a subject suffering from a disorder mediated by PCSK9 expression, the method comprising administering a therapeutically effective amount of an RNAi agent, e.g., a double-stranded RNAi agent, or vector of the present invention to the subject, thereby treating the subject.

[0055] In one embodiment, the subject is a human.

[0056] In one embodiment, the human has hypercholesterolemia.

[0057] In one embodiment, the RNAi agent, e.g., a double-stranded RNAi agent, is administered at a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0058] In one embodiment, the RNAi agent, eg, a double-stranded RNAi agent, is administered subcutaneously or intravenously.

[0059] In one embodiment, the RNAi agent is administered in a dosing regimen including a loading phase followed by a maintenance phase, wherein the loading phase comprises administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg five times per week, and the maintenance phase comprises administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg once, twice, or three times per week, once every two weeks, once every three weeks, once per month, once every two months, once every three months, once every four months, once every five months, or once every six months.

[0060] In one embodiment, the RNAi agent is administered in two or more doses. In certain embodiments, the RNAi agent is administered at intervals selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

[0061] In yet another aspect, the invention provides a method for treating hypercholesterolemia in a subject, the method comprising administering a therapeutically effective amount of an RNAi agent, e.g., a double-stranded RNAi agent, or vector of the invention to the subject, thereby treating the subject.

[0062] In one embodiment, the subject is a primate or rodent, hi another embodiment, the subject is a human.

[0063] In one embodiment, the RNAi agent, e.g., a double-stranded RNAi agent, is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg, hi another embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg.

[0064] In one embodiment, the RNAi agent, eg, a double-stranded RNAi agent, is administered subcutaneously or intravenously.

[0065] In one embodiment, the RNAi agent is administered in a dosing regimen including an initial dosing phase followed by a maintenance phase, wherein the initial dosing phase comprises administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg five times per week, and the maintenance phase comprises administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg once, twice, or three times per week, once every two weeks, once every three weeks, once per month, once every two months, once every three months, once every four months, once every five months, or once every six months.

[0066] In one embodiment, the RNAi agent is administered in two or more doses. In certain embodiments, the RNAi agent is administered at intervals selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

[0067] In one embodiment, the method further comprises determining the subject's LDLR genotype or phenotype.

[0068] In one embodiment, the administration lowers serum cholesterol in the subject.

[0069] In one embodiment, the method further comprises determining serum cholesterol levels in the subject.

[0070] The present invention is further illustrated by the following detailed description and drawings. [Brief explanation of the drawings]

[0071] [Figure 1] 1 is a graph showing that there is a dose-response effect with AD-48400 conjugated to GalNAc at all three doses tested. AD-48399 conjugated to GalNAc serves as a control. [Figure 2A] Graphs showing in vivo efficacy and duration of response for the indicated siRNAs. [Figure 2B]Graphs showing in vivo efficacy and duration of response for the indicated siRNAs. [Figure 3] 1 is a table showing the sequences of the sense (SEQ ID NOS: 1633-1642, respectively, in order of appearance) and antisense (SEQ ID NOS: 1643-1652, respectively, in order of appearance) strands of the duplexes analyzed for in vivo efficacy and lead optimization. [Figure 4] 1 is a graph showing the results of an in vivo efficacy assay for lead optimization. [Figure 5]

[0023] Figure 1 is a graph showing the results of an in vivo dose-response assay performed in PCSK9 transgenic mice. 72 hours after a single dose of 10 mg / kg, 3 mg / kg, 1 mg / kg, and 0.3 mg / kg AD-57928, PCSK9 protein levels were measured by ELISA. [Figure 6] 1 is a graph showing the levels of PCSK9 protein in the serum of PCSK9 transgenic mice after administration of AD-57928 at doses of 5×2 mg / kg during the "initial administration phase" and at doses of 1×2 mg / kg or 2×2 mg / kg during the "maintenance phase." [Figure 7] 1 is a graph showing the levels of PCSK9 protein in the serum of PCSK9 transgenic mice after administration of AD-57928 at doses of 5×1 mg / kg during the "initial administration phase" and at doses of 1×1 mg / kg or 2×1 mg / kg during the "maintenance phase." [Figure 8] 1 is a graph showing the levels of PCSK9 protein in the serum of PCSK9 transgenic mice after administration of AD-57928 at doses of 5×0.5 mg / kg during the "initial administration phase" and at doses of 1×0.5 mg / kg or 2×0.5 mg / kg during the "maintenance phase." [Figure 9]

[0023] Figure 1 is a graph showing the results of an in vivo dose-response assay performed in PCSK9 transgenic mice. 72 hours after a single dose of 0.3 mg / kg siRNA, PCSK9 protein levels were measured by ELISA. [Figure 10]1 is a graph showing the amount of AD-57928 and AD-58895 per nanogram in the liver of C57B6 wild-type mice after administration of a single dose of 1 mg / kg of AD-57928 or AD-58895. [Figure 11] 1 is a graph showing the amount of AD-57928 and AD-58895, expressed as % of the theoretical amount, in the liver of C57B6 wild-type mice after administration of a single dose of 1 mg / kg of AD-57928 or AD-58895. [Figure 12] Figure 12A is a table showing iRNA agents of the invention that include optimized sequences as compared to the AD-57928 sequence. Figure 12A discloses the "sense" sequences as SEQ ID NOS: 1653-1658, respectively, in order of appearance, and the "antisense" sequences as SEQ ID NOS: 1659-1664, respectively, in order of appearance. Figure 12B is a graph showing IC50 values ​​for the indicated iRNA agents. [Figure 13] 1 is a graph showing levels of the indicated iRNA agent in the liver of wild-type mice after administration of a single 1 mg / kg dose of the indicated iRNA agent. [Figure 14] Figure 14A is a graph showing the amount of PCSK9 protein in the serum of non-human primates, expressed as a percentage of PCSK9 remaining relative to pre-bleed levels of PCSK9, after administration of the indicated iRNA agent with qdx5+qwx3. Figure 14B is a graph showing the absolute amount of PCSK9 protein in the serum of non-human primates after administration of the indicated iRNA agent with qdx5+qwx3. [Figure 15] 1 is a graph showing the amount of low density lipoprotein cholesterol (LDL or LDLc) in the serum of non-human primates, expressed as a percent of remaining LDL relative to pre-bleed levels of LDL, following administration of the indicated iRNA agent with qdx5+qwx3. [Figure 16]Figure 16A is a graph showing the amount of low-density lipoprotein cholesterol (LDL or LDLc) in the serum of non-human primates, expressed as a percent of the mean amount of pre-bleed levels of LDL, after administration of AD-57928 at 2 mg / kg q1w and 1 mg / kg 2xw. Figure 16B is a graph showing the amount of PCSK9 protein versus pre-bleed levels in the serum of non-human primates, after administration of AD-57928 at 2 mg / kg q1w and 1 mg / kg 2xw. [Figure 17] Figure 17A is a graph showing the amount of low-density lipoprotein cholesterol (LDL or LDLc) in the serum of non-human primates, expressed as a percent of the mean amount of pre-bleed levels of LDL, after administration of AD-57928 at doses of 2 mg / kg, 2xw, and a single 25 mg / kg. The last dose for the 2 mg / kg, 2xw group was on day 36. Figure 17B is a graph showing the amount of PCSK9 protein relative to the amount pre-bleed in the serum of non-human primates after administration of AD-57928 at doses of 2 mg / kg, 2xw, and a single 25 mg / kg. [Figure 18] 1 is a graph showing the amount of low density lipoprotein cholesterol (LDL or LDLc) in the serum of non-human primates, expressed as a percent of remaining LDL relative to pre-bleed levels of LDL, following administration of the indicated iRNA agent with qdx5+qwx3. [Figure 19] 1 is a graph showing the amount of low density lipoprotein cholesterol (LDL or LDLc) in the serum of non-human primates, expressed as a percent of remaining LDL relative to pre-bleed levels of LDL, following administration of the indicated iRNA agent with qdx5+qwx3. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention provides compositions comprising RNAi agents, e.g., double-stranded iRNA agents, that target PCSK9. Methods of using the compositions of the present invention to inhibit PCSK9 expression and treat pathologies associated with PCSK9 expression, e.g., hypercholesterolemia, are also disclosed.

[0073] I. Definition So that the present invention may be more readily understood, several terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a variable is recited, all values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0074] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0075] The term "including" is used herein to mean, and is used synonymously with, the phrase "including but not limited to."

[0076] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0077] As used herein, "PCSK9" refers to the proprotein convertase subtilisin kexin 9 gene or protein. PCSK9 is also known as FH3, HCHOLA3, NARC-1, or NARC1. The term CSK9 includes human PCSK9 (the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:299523249); mouse PCSK9 (the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:163644257); and rat PCSK9 (the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:77020249). Further examples of PCSK9 mRNA sequences are readily available, for example, using GenBank.

[0078] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of the PCSK9 gene, including mRNA that is the product of RNA processing of a primary transcript.

[0079] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by a sequence given using standard nucleotide nomenclature.

[0080] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" are used interchangeably herein to refer to deoxyribonucleotides in which the nucleobase is thymine, e.g., deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it will be understood that the terms "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to modified nucleotides or surrogate replacement moieties, as described in more detail below. Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted by other moieties without significantly altering the base-pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be substituted in the nucleotide sequences of the invention by nucleotides containing, for example, inosine, and sequences containing such substituted moieties are embodiments of the invention.

[0081] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," as used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as those terms are defined herein. iRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of PCSK9 in cells, e.g., cells in a subject, such as a mammalian subject.

[0082] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, for example, a PCSK9 target mRNA sequence, and induces cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 base pair short interfering RNA with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Then, siRNA is incorporated into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to guide target recognition (Nykanen, et al., (2001) Cell 107:309). When bound to appropriate target mRNA, one or more endonucleases in RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev.15:188). Here, in one aspect, the present invention relates to the single-stranded RNA (siRNA) that is produced in cells and promotes the formation of RISC complex, which leads to the silencing of target gene, i.e., PCSK9 gene. Therefore, the term " siRNA " is also used herein to refer to the above-mentioned RNAi.

[0083] In another embodiment, the RNAi agent can be a single-stranded siRNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs chemically modified as described herein or by the methods described in Lima et al., (2012) Cell 150;:883-894.

[0084] In another embodiment, the "iRNA" for use in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, which are shown to have "sense" and "antisense" orientations relative to the target RNA, i.e., the PCSK9 gene. In one embodiment of the present invention, the double-stranded RNA (dsRNA) causes degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0085] Generally, the majority of the nucleotides in each strand of dsRNA molecule are ribonucleotides; however, as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.Furthermore, as used herein, " RNAi agent " can also contain ribonucleotides with chemical modifications; RNAi agent can contain substantial modifications in multiple nucleotides.Such modifications can include any type of modification disclosed herein or known in the art.Any such modification when used in siRNA type molecules is encompassed by " RNAi agent " for the purpose of this specification and claims.

[0086] The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore connected by a continuous chain of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand, the connected RNA strands are called a "hairpin loop." When the two strands are covalently linked by means other than a continuous chain of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand, the connected structure is called a "linker." The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus the overhang present in the double strand. In addition to the double-stranded structure, the RNAi agent may contain one or more nucleotide overhangs.

[0087] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a PCSK9 target mRNA sequence, and induces cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 base pair short interfering RNA with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of an RNAi agent, when the 3'-end of one strand of the RNAi agent extends beyond the 5'-end of the other strand, or vice versa. "Blunt" or "blunt end" means that there are no unpaired nucleotides at the corresponding end of the double-stranded RNAi agent, i.e., there are no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or RNAi agents with nucleotide overhangs at both ends.

[0088] The term "antisense strand" refers to a strand of a double-stranded RNAi agent (e.g., human PCSK9 mRNA) that contains a region that is substantially complementary to a target sequence. As used herein, the term "region complementary to a portion of mRNA encoding transthyretin" refers to a region of the antisense strand that is substantially complementary to a portion of the PCSK9 mRNA sequence. When the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions, and when present, are generally present in one or more terminal regions, for example, 6, 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0089] The term "sense strand," as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0090] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site in the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage region comprises two bases immediately adjacent to the cleavage site on either side of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0091] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a double-stranded structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under defined conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may occur inside an organism, may also be applied. For example, complementary sequences are sufficient to allow the relevant function of the nucleic acid, such as RNAi, to proceed. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0092] Sequences can be "fully complementary" to each other if there is base pairing between the nucleotides comprising the first nucleotide sequence and the nucleotides comprising the second nucleotide sequence throughout the entire length of the first and second nucleotide sequences.However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences can be completely complementary, or they can form one or more, but generally no more than 4, 3, or 2 mismatched base pairs when hybridized while retaining the ability to hybridize under the conditions most relevant to their final application.However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches when determining complementarity.For example, for the purposes described herein, a dsRNA comprising one oligonucleotide 21 nucleotides long and another oligonucleotide 23 nucleotides long may be referred to as "fully complementary" if the longer oligonucleotide comprises a 21-nucleotide sequence that is completely complementary to the shorter oligonucleotide.

[0093] As used herein, "complementary" sequences can also include, or be formed entirely of, non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairs.

[0094] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to matching bases between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood in the context of their use.

[0095] As used herein, a polynucleotide "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding PCSK9), including the 5' UTR, open reading frame (ORF), or 3' UTR. For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if its sequence is substantially complementary to a contiguous portion of an mRNA encoding PCSK9.

[0096] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0097] As used herein, the phrase "inhibiting the expression of PCSK9" includes inhibiting the expression of any PCSK9 gene (e.g., mouse PCSK9 gene, rat PCSK9 gene, monkey PCSK9 gene, human PCSK9 gene, etc.), as well as variants (e.g., naturally occurring variants) or mutants of the PCSK9 gene. Thus, the PCSK9 gene can be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the context of a genetically modified cell, cell population, or organism.

[0098] "Inhibiting expression of the PCSK9 gene" includes any level of inhibition of the PCSK9 gene, for example, at least partial suppression of expression of the PCSK9 gene, such as at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0099] The expression of the PCSK9 gene can be evaluated based on the level of any variable associated with the expression of the PCSK9 gene, such as the PCSK9 mRNA level, the PCSK9 protein level, or the serum lipid level. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level, or a level measured from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0100] As used herein, the phrase "contacting a cell with a double-stranded RNAi agent" includes contacting a cell by any possible means.Contacting a cell with a double-stranded RNAi agent includes contacting a cell with an RNAi agent in vitro or contacting a cell with an RNAi agent in vivo.Contacting can be performed directly or indirectly.Therefore, for example, an RNAi agent can be physically contacted with a cell by carrying out a method individually, or an RNAi agent can be placed in a situation that allows it to contact a cell later or that allows it to contact a cell later.

[0101] The step of contacting cells in vitro can be carried out, for example, by incubating cells with an RNAi agent.The step of contacting cells in vivo can be carried out, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region, bloodstream, or subcutaneous cavity, so that the RNAi agent can subsequently reach the tissue where the contacted cells are located.For example, the RNAi agent can contain and / or be bound to a ligand, such as a GalNAc3 ligand, that directs the RNAi agent to a target site, for example, the liver.A combination of in vitro and in vivo contact methods is also possible.In connection with the method of the present invention, cells can also be contacted with an RNAi agent in vitro and then transplanted into a subject.

[0102] A "patient" or "subject" as used herein is intended to include either a human or a non-human animal, preferably a mammal, e.g., a monkey. Most preferably, the subject or patient is a human.

[0103] As used herein, "PCSK9-related diseases" is intended to include any disease associated with the PCSK9 gene or protein. Such diseases may be caused, for example, by excessive production of the PCSK9 protein, by mutations in the PCSK9 gene, by abnormal cleavage of the PCSK9 protein, by abnormal interactions between PCSK9 and other proteins, or by other endogenous or exogenous substances. Exemplary PCSK9-related diseases include lipidemia, e.g., hyperlipidemia, and other forms of lipid imbalance, such as hypercholesterolemia, hypertriglyceridemia, and conditions associated with these disorders, such as heart disease and circulatory system disease.

[0104] As used herein, a "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a patient to treat a PCSK9-related disease, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, the method by which the RNAi agent is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by PCSK9 expression, type of previous or concurrent treatment, if any, and other individual characteristics of the patient being treated.

[0105] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject who has not yet developed or exhibited symptoms of a PCSK9-related disease but may be susceptible to the disease. Amelioration of the disease includes delaying the progression of the disease or reducing the severity of later-onset disease. The "prophylactically effective amount" may vary depending on the RNAi agent, the method by which the agent is administered, the risk of developing the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics.

[0106] A "therapeutically effective amount" or "prophylactically effective amount" also includes the amount of an RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNA agents used in the methods of the invention can be administered in amounts sufficient to obtain a reasonable benefit / risk ratio applicable to such treatment.

[0107] As used herein, the term "sample" includes similar body fluids, cells, or tissues isolated from a subject, as well as collections of body fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples can include samples derived from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or body fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma obtained from a subject. In further embodiments, a "sample derived from a subject" refers to liver tissue (or a subcomponent thereof) obtained from a subject.

[0108] II. iRNAs of the Invention Described herein are improved double-stranded RNAi agents and uses of such double-stranded RNAi agents that inhibit expression of the PCSK9 gene in cells, such as cells in a subject, e.g., a mammal (such as a human suffering from a lipid disorder, e.g., hypercholesterolemia).

[0109] Double-stranded RNAi agents of the present invention include, for example, agents having chemical modifications disclosed in U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, the entire contents of which are incorporated herein by reference.

[0110] As shown herein and in U.S. Provisional Patent Application No. 61 / 561,710, better results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand of an RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of an RNAi agent can be completely modified, or alternatively, the introduction of these motifs interrupts the modification pattern of the sense strand and / or antisense strand, if present.The RNAi agent can optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand.The resulting RNAi agent exhibits better gene silencing activity.

[0111] More specifically, it has been surprisingly discovered that when the sense and antisense strands of a double-stranded RNAi agent are fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent, the gene silencing activity of the RNAi agent is significantly improved.

[0112] Thus, the present invention provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., the proprotein convertase subtilisin kexin 9 (PCSK9) gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be in the range of 12 to 30 nucleotides in length. For example, each strand can be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0113] The sense strand and antisense strand typically form double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The double-stranded region of an RNAi agent can be 12 to 30 nucleotide pairs in length. For example, the double-stranded region can be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0114] In one embodiment, an RNAi agent can include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs can form mismatches with the target mRNA, or the overhangs can be complementary to the targeted gene sequence or can be another sequence. The first and second strands can also be joined by additional bases or other non-basic linkers, e.g., to form a hairpin.

[0115] In one embodiment, each nucleotide in the overhang region of an RNAi agent can independently be a modified or unmodified nucleotide, including a 2'-sugar modification, such as, but not limited to, 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be another sequence.

[0116] The 5'- or 3'-overhang on the sense strand, antisense strand, or both strands of an RNAi agent can be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with a phosphorothioate between them, wherein the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0117] RNAi agent can have only one overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand or the 3'-end of the antisense strand.RNAi can also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and the 5'-end is blunt.Without wishing to be bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favors the introduction of the guide strand into the RISC process.

[0118] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 7, 8, and 9, from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0119] In another embodiment, the RNAi agent is a 20-nucleotide long blunt-ended duplex, wherein the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0120] In yet another embodiment, the RNAi agent is a blunt-ended duplex 21 nucleotides in length, wherein the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 9, 10, and 11, from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0121] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt, while the other end comprises two nucleotide overhangs. Preferably, the two nucleotide overhangs are at the 3' end of the antisense strand. When the two nucleotide overhangs are at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the three terminal nucleotides, two of the three nucleotides being overhanging nucleotides, and the third nucleotide being a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in alternating motifs. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0122] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand comprising at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end; the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length such that the RNAi agent reduces expression of the target gene when introduced into a mammalian cell, and dicer cleavage of the RNAi agent preferentially yields siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0123] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs being at the cleavage site of the sense strand.

[0124] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, one of the motifs being at or near the cleavage site on the antisense strand.

[0125] In RNAi agents having a double-stranded region 17-23 nucleotides in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0126] The sense strand of RNAi agent can comprise at least one motif of three identical modifications in three consecutive nucleotides at the break site of strand; antisense strand can have at least one motif of three identical modifications in three consecutive nucleotides at or near the break site of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides in sense strand and one motif of three nucleotides in antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0127] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides. The first motif may be located at or near the cleavage site of the strand, and the other motif may be a wing modification. The term "wing modification" herein refers to a motif located in another part of the strand, away from a motif located at or near the cleavage site of the same strand. The wing modification may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemical structures of the motifs are different from each other; when the motifs are separated by one or more nucleotides, the chemical structures may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be located at one end or on either side of the lead motif relative to the first motif at or near the cleavage site.

[0128] Like the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage. The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present in the sense strand.

[0129] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0130] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0131] When the sense and antisense strands of an RNAi agent each include at least one wing modification, the wing modifications may be located at the same end of the double-stranded region and may have an overlap of 1, 2, or 3 nucleotides.

[0132] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand are each located at one end of the double-stranded region and have an overlap of one, two, or three nucleotides; two modifications from one strand are each located at the other end of the double-stranded region and have an overlap of one, two, or three nucleotides; or two modifications from one strand are located on either side of the lead motif and have an overlap of one, two, or three nucleotides in the double-stranded region.

[0133] In one embodiment, all nucleotides in the sense and antisense strands of an RNAi agent, including nucleotides that are part of a motif, can be modified. Each nucleotide can be modified with the same or different modifications, and these modifications can include one or more changes to one or both of the non-linked phosphate oxygen and / or one or more linking phosphate oxygens; changes to components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; large-scale replacement of the phosphate moiety with a "dephosphorylation" linker; modifications or replacement of natural bases; and replacement or modification of the ribose-phosphate backbone.

[0134] Because nucleic acids are polymers of subunits, many modifications, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties, occur at repeated positions within the nucleic acid. In some cases, modifications can occur at all of the intended positions in the nucleic acid, but often this is not the case. For example, modifications can occur only at the 3' or 5' terminal positions, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in double-stranded and single-stranded regions, especially at the ends. The 5' or both ends can be phosphorylated.

[0135] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide surrogates in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In certain embodiments, all or some of the bases in the 3' or 5' overhang may be modified, e.g., with the modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, e.g., deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribosugar of the nucleobase, according to modifications known in the art, and modifications of the phosphate group, e.g., phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0136] In one embodiment, each residue in the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. A strand may contain two or more modifications. In one embodiment, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0137] At least two different modifications are typically present in the sense and antisense strands, and the two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0138] In one embodiment, N a and / or N b includes an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides in a strand. The alternating nucleotides can refer to one at every other nucleotide or one at every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0139] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0140] In one embodiment, the RNAi agent of the present invention comprises an alternating motif modification pattern in the sense strand that is shifted relative to the alternating motif modification pattern in the antisense strand. This shift can be such that the modification group of the nucleotide of the sense strand corresponds to a different modification group of the nucleotide of the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can start with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BABABA" from 5' to 3' of the strand in the double-stranded region. As another example, the alternating motif in the sense strand can start with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can start with "BBAABBAA" from 5' to 3' of the strand in the double-stranded region, thereby resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0141] In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the sense strand, and this pattern has a first shift with respect to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications in the antisense strand, i.e., the 2'-O-methyl modified nucleotides in the sense strand form base pairs with the 2'-F modified nucleotides in the antisense strand, and vice versa. Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification.

[0142] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand interrupts the original modification pattern present in the sense strand and / or antisense strand. This interruption of the modification pattern of the sense strand and / or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or antisense strand unexpectedly enhances the gene silencing activity against the target gene.

[0143] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different from the modification of the motif. For example, a portion of a sequence containing a motif may be represented by "...N a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications in three consecutive nucleotides, and "N a " and "N b " represents a modification of the nucleotide adjacent to the motif "YYY" that is different from the modification of Y, and N a and N b may be the same or different modifications. a and / or N b may or may not be present if wing modifications are present.

[0144] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may be present at any nucleotide in the sense strand, antisense strand, or both strands, at any position in the strand. For example, the internucleotide bond modification may be present at every nucleotide in the sense strand and / or antisense strand; each internucleotide bond modification may be present in an alternating pattern in the sense strand and / or antisense strand; or the sense strand or antisense strand may contain both internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification in the sense strand may be the same or different from that of the antisense strand, and the alternating pattern of the internucleotide bond modification in the sense strand may have a shift relative to the alternating pattern of the internucleotide bond modification in the antisense strand.

[0145] In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can comprise two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be formed to link the overhang nucleotide with the terminal paired nucleotide in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, there can be additional phosphorothioate or methylphosphonate internucleotide bonds that link the overhang nucleotide with the paired nucleotide adjacent to the overhang nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of the three nucleotides being overhang nucleotides, and the third nucleotide being the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides can be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the antisense strand.

[0146] In one embodiment, the two nucleotide overhangs are at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent can further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0147] In one embodiment, the RNAi agent contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., for the free energy of binding or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With regard to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

[0148] In one embodiment, the RNAi agent includes at least one of the first one, two, three, four, or five base pairs within the double-stranded region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and a mismatch pair, e.g., a non-canonical or non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0149] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0150] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) (In the formula: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; wherein Nb and Y do not have the same modification; XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides. Preferably, all of YYY are 2'-F modified nucleotides.

[0151] In one embodiment, N a and / or N b contains alternating patterns of modifications.

[0152] In one embodiment, the YYY motif is located at or near the cleavage site of the sense strand. For example, if the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the YYY motif can be located at or near the cleavage site of the sense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13).

[0153] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand may be represented by the following formula: 5'n p -Na -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0154] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0155] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0156] When the sense strand is represented as formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0157] Each of X, Y and Z can be the same or different from each other.

[0158] In other embodiments, i is 0, j is 0, and the sense strand may be represented by the formula: 5'n p -N a -YYY-N a -n q 3'(Ia).

[0159] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0160] In one embodiment, the antisense strand sequence of the RNAi has formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3'(II) (In the formula: k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; where N b ' and Y' do not have the same modification; X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides. It can be represented by:

[0161] In one embodiment, N a ' and / or N b ' includes alternating pattern modifications.

[0162] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the Y'Y'Y' motif can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is present at positions 11, 12, or 13.

[0163] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0164] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0165] Thus, the antisense strand can be represented by the following formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb); 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc); or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’3'(IId).

[0166] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0167] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0168] When the antisense strand is represented by formula (IId), each N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0169] In other embodiments, k is 0, l is 0, and the antisense strand may be represented by the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia).

[0170] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0171] Each of X', Y' and Z' can be the same or different from each other.

[0172] Each nucleotide in the sense strand and the antisense strand can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense strand and the antisense strand can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0173] In one embodiment, the sense strand of the RNAi agent may include a YYY motif at positions 9, 10, and 11 of the strand, counting from the first nucleotide from the 5' end if the double-stranded region is 21 nucleotides; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end; Y represents a 2'-F modification. The sense strand may further include a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0174] In one embodiment, the antisense strand may include a Y'Y'Y' motif at positions 11, 12, and 13 of the strand, counting from the first nucleotide from the 5'-end; or optionally, counting from the first paired nucleotide in the double-stranded region from the 5'-end; Y' represents a 2'-O-methyl modification. The antisense strand may further include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0175] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0176] Thus, an RNAi agent for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex has the formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ’ independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; where: Each n may or may not be present p ',n p , n q ', and n q independently represent overhanging nucleotides; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. is expressed by

[0177] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0178] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId)

[0179] When the RNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0180] When the RNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence containing, independently, 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0181] When the RNAi agent is represented as formula (IIIc), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence that independently contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0182] When the RNAi agent is represented as formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b ’ Each of independently comprises an alternating pattern of modifications.

[0183] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) can be the same as or different from each other.

[0184] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides, alternatively, at least two of the Y nucleotides are base-paired with a corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with a corresponding Y' nucleotide.

[0185] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides, or at least two of the Z nucleotides can be base-paired with a corresponding Z' nucleotide; or all three of the Z nucleotides can be base-paired with a corresponding Z' nucleotide.

[0186] When an RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, alternatively, at least two of the X nucleotides are base-paired with the corresponding X' nucleotide; or all three of the X nucleotides are base-paired with the corresponding X' nucleotide.

[0187] In one embodiment, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, and / or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0188] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate bond. a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n pIn another embodiment, when the RNAi agent is represented by formula (IIId), N' is attached to the adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0189] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modification is a 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0190] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0191] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0192] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at one or both of the 5' and 3' ends, and are optionally conjugated to a ligand. Each of the RNAi agents can target the same gene or two different genes; or each of the RNAi agents can target the same gene at two different target sites.

[0193] Various publications describe the multimeric RNAi agent that can be used in the method of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, each of which is incorporated herein by reference in its entirety.

[0194] RNAi agents comprising one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. Ribonucleotide subunits in which the ribose sugar of the subunit has been replaced in this manner are referred to herein as ribose-replacement modified subunits (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0195] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a bond available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, containing a functional group, e.g., a hydroxyl group, or generally a backbone, e.g., a phosphate, or a modified phosphate, e.g., sulfur. In certain embodiments, a "tether attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is attached to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally provide bonds suitable for the incorporation or tethering of another chemical moiety, such as a ligand, to the constituent ring.

[0196] The RNAi agent may be conjugated to the ligand via a carrier, which may be a cyclic group or a cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the cyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0197] In certain embodiments, the RNAi agent for use in the methods of the invention is an agent selected from the group of agents listed in Tables 1 and 2.

[0198] These agents may further comprise a ligand.

[0199] A. Ligand The double-stranded RNA (dsRNA) agent of the present invention can optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the ligand can be conjugated to the sense strand. In a preferred embodiment, the ligand is conjugated to the 3' end of the sense strand. In a preferred embodiment, the ligand is a GalNAc ligand. In a particularly preferred embodiment, the ligand is GalNAc3: [ka]

[0200] In certain embodiments, a ligand, e.g., a GalNAc ligand, is attached to the 3' end of the RNAi agent. [ka] wherein X is O or S. In one embodiment, X is O.

[0201] A variety of components can be attached to the RNAi agents of the invention. Preferred moieties are ligands, which are attached directly or indirectly, preferably covalently, via an intervening tether.

[0202] In preferred embodiments, the ligand alters the distribution, targeting, or lifetime of the molecule into which it is incorporated. In preferred embodiments, the ligand provides improved affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, a receptor, e.g., a cellular or organ compartment, a tissue, an organ, or a region of the body, compared to, e.g., a species in the absence of such ligand. Ligands that provide improved affinity for a selected target are also referred to as targeting ligands.

[0203] Some ligands may have endosomolytic properties. Endosomolytic ligands promote lysis of endosomes and / or transport of the compositions of the present invention, or components thereof, from endosomes to the cytoplasm of cells. Endosomolytic ligands may be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand adopts its active conformation at endosomal pH. An "active" conformation is one in which the endosomolytic ligand promotes lysis of endosomes and / or transport of the compositions of the present invention, or components thereof, from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0204] The ligands can improve the transport, hybridization, and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides, or polymer molecules comprising any combination of the monomers described herein and / or natural or modified ribonucleotides.

[0205] Ligands may generally include therapeutic modifiers, e.g., to enhance uptake; diagnostic compounds or reporter groups, e.g., to monitor distribution; cross-linking agents; and moieties that confer nuclease resistance. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0206] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids, oligonucleotides (e.g., aptamers), and the like. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, and polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, and α-helical peptides.

[0207] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type such as a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptidomimetic, or aptamer.

[0208] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid (cholenic acid), and the like. acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu3+ tetraazamacrocycle conjugate), dinitrophenyl, HRP, or AP.

[0209] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., co-ligands, or antibodies, e.g., molecules with specific affinity for antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. Ligands can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharides, activators of MAP kinase, or activators of NF-κB.

[0210] The ligand can be a substance, e.g., a drug, that can enhance uptake of the iRNA agent into the cell, e.g., by disrupting the cellular microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0211] Ligands can increase oligonucleotide uptake into cells, for example, by activating an inflammatory response. Exemplary ligands that can have such an effect include tumor necrosis factor alpha (TNFα), interleukin-1β, or gamma interferon.

[0212] In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule preferably binds to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to be distributed to target tissues in the body, such as non-renal target tissues. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) adjust the binding to serum proteins, such as HSA.

[0213] Lipid-based ligands can be used to regulate, for example, control, the binding of conjugates to target tissues.For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be removed from the body.Lipids or lipid-based ligands that bind less strongly to HSA can be used to target conjugates to the kidney.

[0214] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity such that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that the HSA-ligand binding cannot be reversed.

[0215] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can also be used in place of or in addition to the lipid-based ligand.

[0216] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful, for example, for treating disorders characterized by unwanted malignant or non-malignant cell proliferation, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low-density lipoproteins (LDL), and high-density lipoproteins (HDL).

[0217] In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic and lipophobic phase.

[0218] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 1). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 2)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 3)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 4)) have been shown to be capable of functioning as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., 2004). al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic attached to the iRNA agent via the incorporated monomer unit is a peptide such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids.The peptide moiety can have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below can be used. An RGD peptide moiety can be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of iRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified, e.g., glycosylated or methylated, to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can target an iRNA agent to a α-. V It can be delivered to tumor cells expressing β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers abundant in proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly cells that display integrins. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides containing D-amino acids, and synthetic RGD mimetics can be used. In addition to RGD, other moieties that target integrin ligands can be used. Generally, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type of ligand target PECAM-1, VEGF, or other oncogenes, such as those described herein.

[0219] A "cell-penetrating peptide" is capable of penetrating cells, e.g., microbial cells such as bacterial or fungal cells, or mammalian cells such as human cells. Peptides that penetrate microbial cells can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0220] In one embodiment, the targeting peptide may be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin. Several factors are believed to preferably maintain the integrity of helical stability. For example, a maximum number of helix-stabilizing residues (e.g., leu, ala, or lys) are used, and a minimum number of helix-destabilizing residues (e.g., proline, or cyclic monomer units) are used. Capping residues are contemplated (e.g., Gly is an exemplary N-capping residue), and / or C-terminal amidation can be used to provide additional H-bonds to stabilize the helix. Stability can be provided by the formation of salt bridges between oppositely charged residues separated by positions i±3, or i±4. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine, or histidine can form salt bridges with the anionic residues glutamic acid or aspartic acid.

[0221] Peptide and peptidomimetic ligands include ligands having natural or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds, i.e., peptide bonds, replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides.

[0222] The targeting ligand can be any ligand that can target a specific receptor. Examples include folate, GalNAc, galactose, mannose, mannose-6P, sugar clusters such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. The ligand can also be based on nucleic acids, for example, aptamers. The aptamer can be unmodified or can have any combination of modifications disclosed herein.

[0223] Endosomal release agents include imidazoles, poly- or oligoimidazoles, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charge, dendrimers with masked or unmasked cationic or anionic charge.

[0224] PK modulators refer to pharmacokinetic modulators. PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention.

[0225] Furthermore, aptamers that bind to serum components (eg, serum proteins) are also suitable as PK-modulating ligands in the present invention.

[0226] Other ligand conjugates suitable for the present invention are described in U.S. patent application Ser. Nos. 10 / 916,185, filed Aug. 10, 2004; 10 / 946,873, filed Sep. 21, 2004; 10 / 833,934, filed Aug. 3, 2007; 11 / 115,989, filed Apr. 27, 2005; and 11 / 944,227, filed Nov. 21, 2007, which are incorporated by reference in their entireties for all purposes.

[0227] When two or more ligands are present, the ligands may all have the same properties, or all have different properties, or some ligands have the same properties while other ligands have different properties.For example, the ligands may have targeting properties, endosomolytic activity, or PK modulating properties.In a preferred embodiment, all ligands have different properties.

[0228] The ligand can be attached to the oligonucleotide at various positions, e.g., the 3' end, the 5' end, and / or an internal position. In a preferred embodiment, the ligand is attached to the oligonucleotide via an intervening tether, e.g., a carrier described herein. The ligand or linking ligand can be present on the monomer as it is incorporated into the growing chain. In some embodiments, the ligand can be incorporated by attachment to a "precursor" monomer after it has been incorporated into the growing chain. For example, a monomer having an amino-terminal tether (i.e., no ligand attached), e.g., TAP-(CH2) nNH can be incorporated into the growing oligonucleotide chain. In a subsequent operation, i.e., after the precursor monomer has been incorporated into the chain, a ligand bearing an electrophilic group, e.g., a pentafluorophenyl ester group or an aldehyde group, can then be attached to the precursor monomer by bonding the electrophilic group of the ligand with the terminal electrophilic group of the tether of the precursor monomer.

[0229] In another example, a monomer bearing a chemical group suitable for participating in a click chemistry reaction can be incorporated into a tether / linker, for example, an azide or alkyne terminated tether / linker. In a subsequent operation, i.e., after the precursor monomer is incorporated into the chain, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer by linking the alkyne and azide together.

[0230] In the case of a double-stranded oligonucleotide, a ligand can be attached to one or both strands. In some embodiments, a double-stranded iRNA agent includes a ligand conjugated to the sense strand. In other embodiments, a double-stranded iRNA agent includes a ligand conjugated to the antisense strand.

[0231] In some embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a nucleic acid molecule. Conjugation to a purine nucleobase or a derivative thereof can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is bound to a conjugate moiety. Conjugation to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to a sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be bound to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be bound to a conjugate moiety, such as in an abasic residue. The internucleoside linkage can also have a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0232] While any suitable ligand in the field of RNA interference can be used, the ligand is typically a carbohydrate, such as a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, or polysaccharide.

[0233] Linkers that conjugate the ligand to the nucleic acid include those described above. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached via a bivalent or trivalent branched linker.

[0234] In one embodiment, the dsRNA of the invention is conjugated to bivalent and trivalent branched linkers comprising the structure shown in any of formulas (IV) to (VII): [ka] During the ceremony: q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5C represents independently for each occurrence 0 to 20, and the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C are each, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently for each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5Care each independently absent for each occurrence, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each, independently for each occurrence, is a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain.

[0235] Trivalent conjugated GalNAc derivatives, such as those of formula (VII), are particularly useful in conjunction with RNAi agents to inhibit expression of target genes: [ka] In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative.

[0236] Examples of suitable divalent and trivalent branched linking groups for conjugation to GalNAc derivatives include, but are not limited to, the following compounds: [ka] [ka] [ka]

[0237] In other embodiments, the RNAi agent for use in the methods of the invention is an agent selected from the group consisting of AD-53815, AD-56663, AD-56658, AD-56676, AD-56666, AD-57928, and AD-60212.

[0238] III. Delivery of iRNA of the Invention Delivery of an iRNA agent of the invention to a cell, e.g., a cell in a subject, such as a human subject (e.g., a subject in need of an iRNA agent, e.g., a subject suffering from a lipid disorder such as dyslipidemia), can be accomplished in several different ways. For example, delivery can be accomplished by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery can also be accomplished directly by administering a composition containing an iRNA, e.g., a dsRNA, to the subject. Alternatively, in vivo delivery can be accomplished indirectly by administering one or more vectors that encode and direct the expression of the iRNA. Examples of these alternatives are described further below.

[0239] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian R.L., (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider when delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, e.g., by direct injection or implantation into the tissue, or by administering the formulation locally. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be adversely affected by or degrade the agent, and can reduce the total dose of the iRNA molecule administered. Several studies have demonstrated successful knockdown of gene products when iRNAs are administered locally. For example, intraocular delivery of VEGF dsRNA via intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al., (2003) Mol. Vis. 9:210-216) have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice can reduce tumor volume (Pille, J. et al., (2005) Mol. Ther. 11:267-274) and prolong the survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523).RNA interference can be delivered locally to the central nervous system by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P.H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G.T. et al. (2004) Neuroscience 129:521-528; Thakker, E.R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y. et al. (2005) J. Neurophysiol. 93:594-602) and locally to the lung by intranasal administration (Howard, K.A. et al. (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55) have also demonstrated success. To administer iRNA systemically for disease treatment, the RNA can be modified or delivered using a drug delivery system; both methods serve to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutical carrier can also enable targeting of iRNA compositions to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to improve cellular uptake and prevent degradation. For example, iRNAs against ApoB conjugated to lipophilic cholesterol moieties were administered systemically to mice, resulting in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, J. et al., (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNAs can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate binding of iRNA molecules (which are negatively charged) and also improve interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNAs or induced to form vesicles or micelles that encapsulate iRNAs (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNAs when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U.N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N. et al. (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S. et al. (2006) Nature 441:111-114), cardiolipin (Chien, P.Y. et al. (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al. (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E. et al. (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D. A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, iRNAs are complexed with cyclodextrins for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

[0240] A. Vector-encoded iRNA of the invention iRNAs targeting the PCSK9 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A. et al., International PCT Publication No. WO 00 / 22113; Conrad, International PCT Publication No. WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a few hours to a few weeks) or sustained (from weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0241] The individual strands of the iRNA can be transcribed from a promoter in an expression vector. Two separate expression vectors can be co-introduced into a target cell (e.g., by transfection or infection) where two separate strands are expressed to produce, for example, dsRNA. Alternatively, each individual strand of the dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence to form a stem-loop structure.

[0242] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing the iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic expression vectors are well known in the art and are available from numerous commercial sources. Such vectors are typically provided containing convenient restriction sites for inserting the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells transplanted from the patient and then reintroduced into the patient, or by any other means that allows for introduction into the desired target cells.

[0243] iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of one week or more are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated using a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using a marker that confers resistance to certain environmental factors (e.g., antibiotics and drugs) on the transfected cells, such as hygromycin B resistance.

[0244] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors, or avian pox, e.g., canarypox or fowlpox, poxvirus vectors; and (j) helper-dependent or attenuated adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may optionally include viral sequences for transfection. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are discussed further below.

[0245] Vectors useful for delivery of iRNA will contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulatable / inducible expression.

[0246] Expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as blood glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0247] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding the iRNAs are cloned into one or more vectors, which facilitate delivery of the nucleic acid to a patient. Further details about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references demonstrating the use of retroviral vectors in gene therapy are Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0248] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155; Mastrangeli et al. (1992), J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94 / 12649; and Wang et al., Gene Therapy 2:775-783 (1995). AV vectors suitable for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0249] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNAs can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101; Fisher KJ et al. (1996), J.Virol, 70:520-532; Samulski R et al. (1989), J.Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0250] Another viral vector suitable for delivery of the iRNA of the invention is a poxvirus, such as a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or an avian pox, such as fowlpox or canarypox.

[0251] The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by engineering the vector to express different capsid protein serotypes. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0252] The pharmaceutical preparation of the vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0253] V. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier is also provided herein. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with PCSK9 gene expression or activity, such as lipid disorders. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral administration, e.g., intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, e.g., by injection into the brain, such as by continuous pump infusion.

[0254] Pharmaceutical compositions containing the RNAi agents of the present invention can be, for example, solutions with or without buffers, or compositions containing pharmaceutically acceptable carriers, including, for example, aqueous or crystalline compositions, liposomal formulations, micelle formulations, emulsions, and gene therapy vectors.

[0255] In the method of the present invention, the RNAi agent can be administered in a solution. The free RNAi agent can be administered in a non-buffered solution, such as saline or water. Alternatively, the free siRNA can also be administered in a suitable buffer. The buffer can contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffer is phosphate-buffered saline (PBS). The pH and osmolality of the buffer containing the RNAi agent can be adjusted to be suitable for administration to a subject.

[0256] In some embodiments, the buffer solution further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, e.g., the physiological value of human plasma. Solutes that can be added to the buffer solution to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0257] The pharmaceutical compositions of the present invention can be administered at a dosage sufficient to inhibit the expression of the PCSK9 gene. Generally, the suitable dose of the iRNA of the present invention is in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally in the range of about 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single administration.

[0258] For example, the RNAi agent, e.g., dsRNA, may have a specific activity 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 contemplated as part of the invention.

[0259] In another embodiment, the RNAi agent, for example, dsRNA, is administered at a concentration 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 mg / kg, about 20 to about 50 mg / kg, about 35 to about 50 mg / kg, about 36 to about 50 mg / kg, about 37 to about 50 mg / kg, about 38 to about 50 mg / kg, about 39 to about 50 mg / kg, about 40 to about 50 mg / kg, about 41 to about 50 mg / kg, about 42 to about 50 mg / kg, about 43 to about 50 mg / kg, about 44 to about 50 mg / kg, about 45 to about 50 mg / kg, about 46 to about 50 mg / kg, about 47 to about 50 mg / kg, about 48 to about 50 mg / kg, about 49 to about 50 mg / kg, about 50 to about 50 mg / kg, about 51 to about 51 mg / kg, about 52 to about 52 mg / kg, about 53 to about 53 mg / kg, about 54 to about 54 mg / kg, 0mg / kg, about 20 to about 50mg / kg, about 25 to about 50mg / kg, about 25 to about 50mg / kg, about 30 to about 50mg / kg, about 35 to about 50mg / kg, about 40 to about 50mg / kg, about 45 to about 50mg / kg, about 0.1 to about 45mg / kg, about 0.25 to about 45mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 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 4 5mg / kg, about 35 to about 45mg / kg, about 40 to about 45mg / kg, about 0.1 to about 40mg / kg, about 0.25 to about 40mg / kg, about 0.5 to about 40mg / kg, about 0.75 to about 40mg / kg, about 1 to about 40mg / mg, about 1.5 to about 40mg / kb, about 2 to about 40m g / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, About 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m g / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present invention.

[0260] For example, an RNAi agent, e.g., dsRNA, may have a specific activity 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, 5.0, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19, 5.20, 5.21, 5.22, 5.23, 5.24, 5.25, 5.26, 5.27, 5.28, 5.29, 5.30, 5.31, 5.32, 5.33, 5.34, 5.35, 5.36, 5.37, 5.38, 5.39, 5.40, 5. The compound may be administered at a dose of 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0261] In another embodiment, the RNAi agent, e.g., dsRNA, is administered at a concentration of about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, 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 25 to about 50 mg / kg, or about 30 to about 50 mg / kg. 0mg / kg, about 25 to about 50mg / kg, about 30 to about 50mg / kg, about 35 to about 50mg / kg, about 40 to about 50mg / kg, about 45 to about 50mg / kg, about 0.5 to about 45mg / kg, about 0.75 to about 45mg / kg, about 1 to about 45mg / mg, about 1.5 to about 45m g / 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 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 0.7 5 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 m g / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg , about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / k g, 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 The dsRNA is administered at a dose of 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. In one embodiment, the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the present invention.

[0262] For example, the subject may be administered approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19, 5.20, 5.21, 5.22, 5.23, 5.24, 5.25, 5.26, 5.27, 5.28, 5.29, 5.30, 5.31, 5.32, 5.33, 5.34, 5.35, 5.36, 5.37, 5.38, 5.39, 5.40, 5.41, 5.42, 5.43, 5.44, 5.45, 5.46, 5.47, 5.48, 5.49, 5.50, 5.51, 5.52, 5.53, 5.54, 5.55, 5.5 ,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 ,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16.5,17,17.5,18,18.5,19,19.5,20,20.5,21,21.5,2 Therapeutic amounts of iRNA such as 2, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg may be administered. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0263] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more subdoses at appropriate intervals throughout the day, or even via continuous infusion or delivery via sustained-release formulations. In such cases, each subdose must contain a correspondingly smaller amount of iRNA to achieve the total daily dosage. The dosage unit can also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of the iRNA over a period of several days. Sustained-release formulations are well known in the art and can be used with the agents of the present invention, as they are particularly useful for delivering agents to specific sites. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0264] In other embodiments, a single dose of the pharmaceutical composition can be administered prolonged, with subsequent doses administered at intervals of no more than 3, 4, or 5 days, or no more than 1, 2, 3, or 4 weeks. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered weekly. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered bi-monthly.

[0265] Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can influence the dosage and duration required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for individual iRNAs encompassed by the invention can be estimated using conventional methodology or based on in vivo testing using appropriate animal models, as described elsewhere herein.

[0266] Advances in mouse genetics have produced many mouse models for the study of various human diseases, such as bleeding disorders, which can benefit from the reduction of PCSK9 expression.Such models can be used for the in vivo testing of iRNA and for determining therapeutically effective doses.Suitable mouse models are known in the art, and include, for example, the mouse that contains the transgene that expresses human PCSK9.

[0267] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is required and the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary administration, e.g., by inhalation or insufflation of powders or aerosols, such as with a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous administration, e.g., via an implanted device; or intracranial administration, e.g., via intraparenchymal, intrathecal, or intraventricular administration.

[0268] The iRNA can be delivered to target a specific tissue, such as the liver (e.g., hepatocytes of the liver).

[0269] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is mixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleyltetramethylaminopropyl DOTAP and dioleylphosphatidylethanolamine DOTMA). The iRNAs featured in the present invention can be encapsulated in liposomes or complexed to liposomes, particularly cationic liposomes. Alternatively, the iRNAs can be complexed to lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

[0270] A. iRNA formulations containing membrane molecular assemblies iRNAs for use in the compositions and methods of the present invention can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar and multilamellar vesicles, whose membrane is formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior and typically does not contain the iRNA composition, although in some cases it may. Liposomes are useful for transporting and delivering active ingredients to sites of action. Because the liposome membrane is structurally similar to biological membranes, when the liposome is attached to a tissue, the liposome bilayer fuses with the cell membrane bilayer. As the liposome and cell fuse, the aqueous contents, including the iRNA, are delivered to the cell, where the iRNA can specifically bind to the target RNA and mediate RNAi. In some cases, the liposomes are also specifically targeted, for example, to direct the iRNA to a particular cell type.

[0271] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent so that micelles are formed with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. Next, a preparation of the RNAi agent is added to the micelles containing the lipid components. The cationic groups in the lipids interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of the RNAi agent.

[0272] If necessary, a carrier compound that aids in condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to aid in condensation.

[0273] Methods for producing stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., M. Mol. al.,Biochim.Biophys.Acta 557:9,1979;Szoka et al.,Proc.Natl.Acad.Sci.75:4194,1978;Mayhew et al.,Biochim.Biophys.Acta 775:169,1984;Kim et al.,Biochim.Biophys.Acta 728:339,1983; and Fukunaga et al. The present invention may also include one or more embodiments of the exemplary method described in

[0010] et al., Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of an appropriate size for use as a delivery vehicle include sonication and freeze-thaw and extrusion (see, e.g., Mayer et al., Biochim. Biophys. Acta 858:161, 1986). When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., Biochim. Biophys. Acta 775:169, 1984). These methods are easily adapted to packaging preparations of RNAi agents into liposomes.

[0274] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are transported into endosomes. The acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0275] pH-sensitive, negatively charged liposomes entrap nucleic acids rather than complexing them. Because both the nucleic acid and the lipid are similarly charged, repulsion occurs rather than complexation. Nevertheless, some nucleic acids are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0276] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic membrane-fusogenic liposomes are primarily formed from dioleylphosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0277] Other exemplary methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss, EMBO J. 11:417, 1992.

[0278] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. The results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A in different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4(6)466).

[0279] Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) those that are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that the enhanced circulation half-life of these sterically stabilized liposomes, at least for those containing gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0280] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported the use of monosialoganglioside G, which improves the blood half-life of liposomes. M1 reported the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. These findings are detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0281] In one embodiment, cationic liposome is used.Cationic liposome has the advantage that it can fuse with cell membrane.Non-cationic liposome cannot fuse with cell membrane as efficiently, but it can be taken up by macrophage in vivo and can be used to deliver RNAi agent to macrophage.

[0282] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; and liposomes can protect the RNAi agent encapsulated in their internal compartment from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size and aqueous volume of the liposome.

[0283] A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids, forming lipid-nucleic acid complexes that can fuse with the negatively charged lipids of tissue culture cell membranes, resulting in delivery of RNAi agents (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0284] DOTMA analogue, 1,2-bis(oleyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously adhere to negatively charged cell surfaces, fuse with the cell membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is attached by an ester rather than an ether bond.

[0285] Other reported cationic lipid compounds include those conjugated to one of two types of lipids and conjugated to various moieties, including, for example, carboxyspermine, including compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

[0286] Another cationic lipid conjugate involves derivatizing lipids with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, prepared by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0287] Liposome preparations are particularly suitable for topical administration, and liposomes have several advantages over other preparations.These advantages include the reduction of side effects associated with the high systemic absorption rate of administered drug, the increase in the accumulation of administered drug in desired target, and the ability to administer RNAi agent to skin.In some implementations, liposomes are used to deliver RNAi agent to epidermal cells and promote the penetration of RNAi agent into dermal tissue, for example, skin.For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been reported (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992:259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al., Gene 56:267-276, 1987; Nicolau, C. et al. (1987) Meth. Enz. 149:157-176, 1987; Straubinger, R M and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C Y and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0288] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in delivering drugs to the skin. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing RNAi agents are useful for treating skin diseases.

[0289] Liposomes containing iRNA can be made highly deformable. Such deformability can allow the liposomes to pass through pores smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be made by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing RNAi agents can be delivered, for example, by subcutaneous injection to deliver the RNAi agent to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to their lipid properties, these transferosomes can be self-optimizing (e.g., adaptable to the shape of pores), self-repairing, often reaching their target without fracture, and often self-loading.

[0290] Other formulations suitable for the present invention are described in U.S. Provisional Patent Applications Nos. 61 / 018,616, filed January 2, 2008; 61 / 018,611, filed January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008; and 61 / 051,528, filed May 8, 2008. PCT Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations suitable for the present invention.

[0291] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can also be described as lipid droplets, which, because of their high deformability, can easily penetrate pores smaller than the droplets. Transfersomes can adapt to the environment in which they are used, for example, they are self-optimizing (adapting to the shape of pores in the skin), self-repairing, often reach their targets without fragmentation, and are often self-loading. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0292] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0293] If the surfactant molecule is not ionized, the surfactant is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0294] When surfactant molecule dissolves or disperses in water, if it carries negative charge, this surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

[0295] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic.Cationic surfactants include quaternary ammonium salts and ethoxylated amines.Quaternary ammonium salts are the most commonly used members of this class.

[0296] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0297] The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0298] The iRNA for use in the method of the present invention can also be provided as a micelle formulation. " Micelle " is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure, with all hydrophobic parts of the molecule facing inward, and the hydrophilic parts remaining in contact with the surrounding aqueous phase. When the environment is hydrophobic, the opposite arrangement exists.

[0299] Mixed micelle formulations suitable for transdermal delivery contain an aqueous solution of the siRNA composition, alkali metal C8-C 22 They can be prepared by mixing alkyl sulfates and micelle-forming compounds. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compounds may be added simultaneously with or after the addition of alkali metal alkyl sulfates. Mixed micelles can be formed by virtually any type of mixing of components, but more vigorous mixing is preferred to provide smaller micelles.

[0300] In one method, a first micelle composition containing an siRNA composition and at least an alkali metal alkyl sulfate is prepared. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, the micelle composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulfate, and at least one of the micelle-forming compounds, followed by adding the remaining micelle-forming compounds while vigorously mixing.

[0301] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0302] To deliver a micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, which is then filled with a propellant. The propellant, under pressure, is in liquid form in the dispenser. The ratio of the components is adjusted so that there is one aqueous phase and one propellant phase, i.e., one phase. If two phases are present, the dispenser must be shaken before dispensing a portion of its contents, for example, via a metered valve. The medicinal dose is then expelled from the metered valve in the form of a fine spray.

[0303] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

[0304] The specific concentrations of the essential components can be determined by relatively simple experimentation. For absorption via the oral cavity, it is often desirable to increase the dosage, for example, by at least two or three times, that for injection or administration via the gastrointestinal tract.

[0305] B. Lipid particles The iRNA or dsRNA of the present invention may be fully encapsulated in a lipid formulation, such as an LNP, or may form other nucleic acid-lipid particles.

[0306] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs have an extended circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them extremely useful for systemic applications. LNPs include "pSPLPs," which contain encapsulated condensing agent-nucleic acid complexes, as described in PCT Publication No. WO 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Additionally, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and PCT Publication No. WO 96 / 40964.

[0307] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above ranges are also considered part of the invention.

[0308] Examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-di Methylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof , (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0309] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0310] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mol percent), and have a particle size of 63.0±20 nm and a 0.027 siRNA / lipid ratio.

[0311] Ionic / non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N The lipid may be an anionic or neutral lipid, including 1-(2-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid, when cholesterol is included, may comprise about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0312] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol%, or 2 mol% of the total lipid present in the particle.

[0313] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0314] In one embodiment, lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each can be prepared in ethanol as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-Ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., in sodium acetate (pH 5)) so that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles usually form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder such as the Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate-buffered saline (PBS), for example, at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. [ka]

[0315] LNP01 formulations are described, for example, in International Application Publication No. WO 2008 / 042973, which is incorporated herein by reference.

[0316] Further exemplary lipid-dsRNA formulations are described in Table A.

[0317] [Table 1]

[0318] [Table 2]

[0319] DSPC: Distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000) Formulations containing LNP (l,2-dilinolenyloxy-N,N-dimethylaminepropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0320] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application Nos. 61 / 148,366, filed January 29, 2009; 61 / 156,851, filed March 2, 2009; 61 / 156,851, filed June 10, 2009; 61 / 228,373, filed July 24, 2009; 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0321] Formulations containing MC3 are described, for example, in US Patent Application Publication No. 2010 / 0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference.

[0322] Formulations containing ALNY-100 are described, for example, in International Patent Application No. PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0323] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0324] Synthesis of ionic / cationic lipids Any of the compounds, e.g., cationic lipids, used in the nucleic acid-lipid particles of the present invention can be prepared by known organic synthesis techniques, including those methods described in more detail in the Examples. All substituents are as defined below unless otherwise indicated.

[0325] "Alkyl" means a straight-chain or branched-chain, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched-chain alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0326] "Alkenyl" refers to an alkyl as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight and branched chain alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0327] "Alkynyl" means any alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative straight and branched chain alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0328] "Acyl" means any alkyl, alkenyl, or alkynyl group in which the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0329] "Heterocycle" means a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocycle that is either saturated, unsaturated, or aromatic and contains one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, where the nitrogen and sulfur heteroatoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be bonded via any heteroatom or carbon atom. Heterocycle includes heteroaryl, as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0330] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle," when substituted, mean that at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, -CN, -ORx, -NRxRy, -NRxC(=O)Ry, -NRxS02Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy, where n is 0, 1 or 2 and Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORx, heterocycle, -NRxRy, -NRxC(=O)Ry, -NRxS02Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy.

[0331] "Halogen" means fluoro, chloro, bromo and iodo.

[0332] In some embodiments, the methods of the present invention may require the use of protecting groups. Protecting group methods are well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, TW et al., Wiley-Interscience, New York City, 1999). Briefly, a protecting group in the context of the present invention is any group that reduces or eliminates the undesired reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during a specific reaction and then removed to reveal the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the undesired reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0333] Synthesis of Formula A In one embodiment, the nucleic acid-lipid particles of the present invention have Formula A: [ka] In some embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Generally, the lipid of formula A above can be prepared by the following reaction scheme 1 or 2, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 can be taken together to form an optionally substituted heterocycle.

[0334] Scheme 1 [ka] Lipids A (wherein R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be optionally substituted; R3 and R4 are independently lower alkyl; or R3 and R4 can be joined to form an optionally substituted heterocycle) can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 provides ketal 3. Treatment of ketal 3 with amine 4 provides lipids of formula A. Lipids of formula A can be converted to the corresponding ammonium salt using an organic salt of formula 5 (wherein X is an anionic counterion selected from halogen, hydroxide, phosphate, sulfate, etc.).

[0335] Scheme 2 [ka] Alternatively, the starting material ketone 1 can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 6 and 7 gives ketone 1. Conversion of ketone 1 to the corresponding lipid of formula A is depicted in Scheme 1.

[0336] Synthesis of MC3 DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was prepared as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid and then with dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed on a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. Fractions containing the purified product were combined. The solvent was removed to give a colorless oil (0.54 g). Synthesis of ALNY-100

[0337] The synthesis of ketal 519 [ALNY-100] was carried out according to Scheme 3: [ka]

[0338] Synthesis of 515 To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mL of anhydrous THF in a two-necked round-bottom flask (1 L) was slowly added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF at 0°C under a nitrogen atmosphere. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. The progress of the reaction was monitored by TLC. After completion of the reaction (by TLC), the mixture was cooled to 0°C and quenched by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 h and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. The volatiles were removed under reduced pressure to give the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1 H-NMR (DMSO, 400MHz): δ = 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66~2.60 (m, 2H), 2.50~2.45 (m, 5H).

[0339] Synthesis of 516 To a stirred solution of compound 515 in 100 mL of dry DCM in a 250 mL two-neck round-bottom flask, NEt3 (37.2 mL, 0.2669 mol) was added and cooled to 0 °C under a nitrogen atmosphere. N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM was slowly added, and the reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC), the mixture was washed successively with 1 N HCl solution (1 × 100 mL) and saturated NaHCO3 solution (1 × 50 mL). The organic layer was then dried over anhydrous Na2SO4, and the solvent was evaporated to give crude material, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400MHz): δ=7.36~7.27(m,5H), 5.69(s,2H), 5.12(s,2H), 4.96(br.,1H)2.74(s,3H), 2.60(m,2H), 2.30~2.25(m,2H). LC-MS[M+H]-232.3(96.94%).

[0340] Synthesis of 517A and 517B Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of a 10:1 solution of acetone and water in a 500 mL single-neck round-bottom flask, and N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added thereto, followed by 4.2 mL of a 7.6% solution of OsO (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (approximately 3 h), the mixture was quenched by the addition of solid NaSO, and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over anhydrous NaSO, and the solvent was removed under reduced pressure. Silica gel column chromatography purification of the crude material afforded a mixture of diastereomers, which were separated by preparative HPLC. Yield: 6 g of crude 517A-Peak-1 (white solid), 5.13 g (96%). H-NMR (DMSO, 400 MHz): δ = 7.39–7.31 (m, 5H), 5.04 (s, 2H), 4.78–4.73 (m, 1H), 4.48–4.47 (d, 2H), 3.94–3.93 (m, 2H), 2.71 (s, 3H), 1.72–1.67 (m, 4H). LC-MS: [M+H] = 266.3, [M+NH] = 283.5 present, HPLC = 97.86%. Stereochemistry confirmed by X-ray.

[0341] Synthesis of 518 Using a procedure similar to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. H-NMR (CDCl, 400 MHz): δ = 7.35–7.33 (m, 4H), 7.30–7.27 (m, 1H), 5.37–5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58–4.57 (m, 2H), 2.78–2.74 (m, 7H), 2.06–2.00 (m, 8H), 1.96–1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37–1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

[0342] General procedure for the synthesis of compound 519 A solution of compound 518 (1 equiv.) in hexane (15 mL) was added dropwise to an ice-cooled solution of LAH in THF (1 M, 2 equiv.). After complete addition, the mixture was heated at 40° C. for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous NaSO, then filtered through Celite and reduced to an oil. Column chromatography afforded pure 519 (1.3 g, 68%), which was obtained as a colorless oil. 13 C NMR δ = 130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; electrospray MS (+ve): C 44 H 80 Molecular weight (M+H) for NO2 calculated 654.6, found 654.6.

[0343] Formulations prepared by either standard or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. The formulation should be a whitish, translucent solution without aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be approximately 20-300 nm in size, e.g., 40-100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation and the entrapped fraction can be estimated using a dye exclusion assay. A sample of the formulated dsRNA may be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by comparing the signal from the sample containing the surfactant against a standard curve. The entrapped fraction is determined by subtracting the "free" dsRNA content (measured by the signal in the absence of surfactant) from the total dsRNA content. The percent of entrapped dsRNA is typically >85%. For LNP formulations, particle sizes are at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Suitable ranges are typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0344] Oral compositions and formulations include powders or granules, microparticles, nanoparticles, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be desirable. In some embodiments, oral formulations are those in which the dsRNA characterizing the present invention is administered with one or more permeation enhancers, surfactants, and chelating agents. Suitable surfactants include fatty acids and / or esters or their salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as a fatty acid / salt in combination with a bile acid / salt. An exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA characterizing the present invention can be orally delivered in the form of granules, including spray-dried particles, or complexed to form micro- or nanoparticles.dsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkylcyanoacrylates, DEAE-derivatized polyimines, pullulan, cellulose, and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate. Oral formulations for dsRNA and their formulations are described in U.S. Pat. No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0345] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0346] The pharmaceutical compositions of the present invention include, but are not limited to, liquids, emulsions, and liposome-containing formulations. These compositions can be made from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases such as hepatocarcinoma, liver-targeting formulations are particularly preferred.

[0347] The pharmaceutical formulations of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with pharmaceutical carriers or excipients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0348] The compositions of the present invention can be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer.

[0349] C. Further Formulations i. Emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets, usually greater than 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and (See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w) types. When an aqueous phase is finely divided and dispersed as minute droplets into a bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when an oil phase is finely divided and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.In addition to the dispersed phase and active drug, emulsions can contain additional components, which may be present in the aqueous phase, as a solution in the oil phase, or as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants can also be present in the emulsion as needed. Pharmaceutical emulsions can be multiple emulsions consisting of three or more phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o / w emulsion surround small water droplets constitute w / o / w emulsions. Similarly, a system of oil droplets surrounded by globules of water stabilized in a continuous oil phase provides an o / w / o emulsion.

[0350] Emulsions are characterized by having little or no thermodynamic stability.In many cases, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase and is maintained in this form through the use of emulsifiers or the viscosity of the formulation.Either phase of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams.Other means of stabilizing emulsions include the use of emulsifiers, which can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0351] Synthetic surfactants, also known as surface active agents, have found widespread application in the formulation of emulsions and have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199). Surfactants are usually amphiphilic, comprising a hydrophilic portion and a hydrophobic portion. The ratio of hydrophilicity to hydrophobicity of surfactant is called hydrophilic / lipophilic balance (HLB), which is a valuable tool for classifying and selecting surfactants when preparing formulations.Surfactants can be classified into different types based on the nature of hydrophilic groups, namely, nonionic, anionic, cationic and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285).

[0352] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases, such as anhydrous lanolin and hydrophilic petrolatum, possess hydrophilic properties that allow them to incorporate water to form water-in-oil emulsions while still maintaining their semisolid consistency. Finely divided solids have been used as good emulsifiers, especially in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0353] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions, including fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0354] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around droplets of the dispersed phase and by increasing the viscosity of the external phase.

[0355] Emulsions often contain many ingredients, such as carbohydrates, proteins, sterols, and phosphatides, which can easily support the growth of microorganisms, so these preparations often incorporate preservatives. Commonly used preservatives in preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also usually added to emulsion preparations to prevent the preparation from deteriorating. The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0356] The application of emulsion formulations via the dermal, oral and parenteral routes, as well as their manufacturing methods, have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of formulation and effectiveness in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional formulations are among the materials commonly administered orally as oil-in-water emulsions.

[0357] ii. Microemulsions In one embodiment of the present invention, iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphile that is a single, optically isotropic, and thermodynamically stable liquid solution (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution, followed by the addition of a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a clear system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared using a combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in: Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0358] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with extensive knowledge of how to formulate microemulsions (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that form spontaneously.

[0359] Surfactants used in preparing microemulsions, alone or in combination with cosurfactants, include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750). The cosurfactant, typically a short-chain alcohol such as ethanol, 1-propanol, or 1-butanol, serves to increase interfacial fluidity by penetrating the surfactant film, resulting in the formation of an irregular film due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include materials such as, but not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0360] Microemulsions are particularly interesting from the standpoint of drug solubilization and drug absorption enhancement. Lipid-based microemulsions (both o / w and w / o) have been proposed to improve the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105, 7,063,860, 7,070,802, 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105, 7,063,860, 7,070,802, 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can form spontaneously when the components of the microemulsion are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or iRNA. Microemulsions are also effective for the efficient delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improved local cellular uptake of iRNA and nucleic acids.

[0361] The microemulsions of the present invention may also contain additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve formulation properties and enhance absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0362] iii. Particulates The RNAi agents of the present invention may be incorporated into particles, such as microparticles. Microparticles can be produced by spray drying, but can also be produced by other methods, including freeze-drying, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

[0363] iv. Penetration enhancers In one embodiment, the present invention utilizes various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to the skin of animals. Most drugs exist in solution in both ionized and non-ionized forms. However, typically, only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the permeability of lipophilic drugs.

[0364] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above types of penetration enhancers is described in more detail below.

[0365] Surfactants (or "surface-active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of iRNA through mucosal membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0366] Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1~20 Included are alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their monoglycerides and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, for example, Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0367] The physiological role of bile includes facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee, et al.,Critical Reviews in Therapeutic Drug Carrier Systems,1991,page 92;Swinyard,Chapter 39 In:Remington's Pharmaceutical Sciences,18th Ed.,Gennaro,ed.,Mack Publishing Co.,Easton,Pa.,1990,pages 782-783;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0368] Chelators used in connection with the present invention can be defined as compounds that remove metal ions from solution by forming complexes with the metal ions, resulting in enhanced absorption of iRNA through mucosal membranes. With regard to the use of chelators as penetration enhancers in the present invention, chelators have the added advantage of also acting as DNase inhibitors, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelators (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives (enamines) of β-diketones (see, for example, Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0369] As used herein, non-chelating, non-surfactant penetration enhancers can be defined as compounds that exhibit minimal activity as chelators or surfactants but nonetheless enhance the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of penetration enhancers of this type include unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacycloalkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 92); and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0370] Agents that enhance cellular uptake of iRNA can also be added to pharmaceutical and other compositions of the present invention. For example, cationic lipids such as lipofectin (Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., PCT Application WO 97 / 30731) are known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), RNAiMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;(Madison, WI), TransFast (trademark) Transfection Reagent (Promega; Madison, WI), Tfx (trademark)-20 Reagent (Promega; Madison, WI), Tfx (trademark)-50 Reagent (Promega; Madison, WI), DreamFect (trademark) (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass; aD1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM) / LipoGen(TM) (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER(TM) transfection Reagent (Genlantis; San Examples of suitable fusion proteins include RiboFect (Bioline; Taunton, MA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), and HiFect™ (B-Bridge International, Mountain View, CA, USA).

[0371] Other agents can be used to enhance penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azone, and terpenes such as limonene and menthone.

[0372] v. Carrier Certain compositions of the present invention also incorporate a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or analog thereof that may be inert (i.e., not possessing biological activity) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or promoting its removal from the circulation. Co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter, can substantially reduce the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, possibly due to competition between the carrier compound and the nucleic acid for a common receptor. For example, recovery of partial phosphorothioates in liver tissue can be reduced when they are co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0373] vi. excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected to provide the desired bulk, consistency, etc. when combined with the nucleic acid and other desired components of the pharmaceutical composition, taking into account the planned method of administration. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); tablet disintegrants (such as starch, sodium starch glycolate, and the like); and wetting agents (such as sodium lauryl sulfate, and the like).

[0374] Suitable organic or inorganic excipients that are pharmaceutically acceptable for parenteral administration and do not adversely react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0375] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions, or solutions of nucleic acids in liquid or solid oil bases in common solvents such as alcohol. Solutions can also contain buffers, diluents, and other suitable additives. Suitable organic or inorganic excipients that are pharmaceutically acceptable for parenteral administration and do not adversely react with nucleic acids can be used.

[0376] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0377] vii. Other components The compositions of the present invention may also contain other auxiliary components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically active ingredients, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, these materials should not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if desired, mixed with auxiliary substances that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0378] The aqueous suspension may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer.

[0379] In some embodiments, pharmaceutical compositions featured in the present invention comprise (a) one or more iRNA compounds and (b) one or more agents that function via a non-RNAi mechanism and are useful for treating bleeding disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-steatotic agents, antiviral agents, and / or anti-fibrotic agents. Additionally, other substances commonly used to protect the liver, such as silymarin, can also be used with the iRNAs described herein. Other agents useful for treating liver disease include protease inhibitors such as telbivudine, entecavir, and telaprevir, as well as other agents disclosed, for example, in U.S. Patent Application Publications 2005 / 0148548, 2004 / 0167116, and 2003 / 0144217 to Tung et al.; and U.S. Patent Application Publication 2004 / 0127488 to Hale et al.

[0380] The toxicity and therapeutic efficacy of such compounds are discussed, for example, in the LD 50 (lethal dose for 50% of the population) and ED 50 The LD (the dose therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Compounds that exhibit high therapeutic indices are preferred.

[0381] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosages of the compositions featured herein in the present invention are generally administered with little or no toxicity to the ED 50The dosage may vary within this range depending on the dosage form and route of administration employed. For any compound used in the methods featured in the present invention, a therapeutically effective dose can be initially estimated from cell culture assays. A dose can be formulated to achieve a circulating plasma concentration range of the compound, or, if appropriate, the polypeptide product of the target sequence (e.g., achieve a reduction in polypeptide concentration), in an animal model that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as measured in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0382] In addition to those administrations described above, the iRNAs featured in the present invention may be administered in combination with other known agents effective in treating pathological processes mediated by PCSK9 expression. In either case, the administering physician can adjust the amount and time of administration of the iRNA based on the observed results, using standard efficacy measures known in the art or described herein.

[0383] IV. Methods for Inhibiting PCSK9 Expression The present invention provides a method for inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, comprising contacting the cell with an RNAi agent, e.g., a double-stranded RNAi agent, in an amount effective to inhibit expression of PCSK9 in the cell, thereby inhibiting expression of PCSK9 in the cell.

[0384] The step of contacting a cell with a double-stranded RNAi agent can be carried out in vitro or in vivo. The step of contacting a cell with an RNAi agent in vivo includes contacting a cell or a group of cells in a subject, for example, a human subject, with the RNAi agent. A combination of in vitro and in vivo contacting methods is also possible. The contacting can be carried out directly or indirectly, as described above. Furthermore, the step of contacting a cell can be carried out by a targeting ligand, including any ligand described herein or known in the art. In a preferred embodiment, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand, or any other ligand that directs the RNAi agent to a target site, for example, the liver of a subject.

[0385] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," and other similar terms, and includes any level of inhibition.

[0386] As used herein, the phrase "inhibiting the expression of PCSK9" is intended to refer to the inhibition of the expression of any PCSK9 gene (such as a mouse PCSK9 gene, a rat PCSK9 gene, a monkey PCSK9 gene, or a human PCSK9 gene), as well as variants or mutants of the PCSK9 gene. Thus, the PCSK9 gene may be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the context of a genetically modified cell, cell group, or organism.

[0387] "Inhibiting expression of the PCSK9 gene" includes any level of inhibition of the PCSK9 gene, for example, at least partial suppression of expression of the PCSK9 gene. Expression of the PCSK9 gene can be assessed based on the level or change in level of any variable associated with expression of the PCSK9 gene, for example, PCSK9 mRNA level, PCSK9 protein level, or lipid level. The level can be assessed, for example, in an individual cell or a group of cells, including a sample derived from a subject.

[0388] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables related to PCSK9 expression compared to a control level, which can be any type of control level used in the art, such as a pre-dose baseline level or a level measured from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control, etc.).

[0389] In certain embodiments of the methods of the present invention, expression of the PCSK9 gene is inhibited 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 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%.

[0390] Inhibition of expression of the PCSK9 gene is manifested by a reduction in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) that has been treated so that the PCSK9 gene is transcribed and expression of the PCSK9 gene is inhibited (e.g., by contacting one or more cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present), compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been so treated. In a preferred embodiment, inhibition is assessed by expressing the level of mRNA in the treated cells as a percentage of the level of mRNA in the control cells using the following formula:

number

[0391] Alternatively, inhibition of PCSK9 gene expression can be evaluated in terms of a decrease in a parameter functionally related to PCSK9 gene expression, for example, PCSK9 protein expression, such as lipid level, cholesterol level, for example, LDLc level. PCSK9 gene silencing can be measured in any cell that expresses PCSK9, structurally or by genome engineering, and by any assay known in the art. The liver is the main site of PCSK9 expression. Other substantial sites of expression include the pancreas, kidney, and intestine.

[0392] Inhibition of PCSK9 protein expression is manifested by a decrease in the level of PCSK9 protein expressed by a cell or group of cells (e.g., the level of the protein expressed in a sample derived from a subject). As described above for assessing mRNA suppression, inhibition of the expression level of the protein in a treated cell or group of cells can similarly be expressed as a percentage of the level of the protein in a control cell or group of cells.

[0393] Control cells or cell populations that can be used to assess inhibition of PCSK9 gene expression include cells or cell populations that have not yet been contacted with an RNAi agent of the present invention. For example, control cells or cell populations are obtained from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

[0394] The level of PCSK9 mRNA expressed by a cell or group of cells can be measured using any method known in the art for assessing mRNA expression. In one embodiment, the level of PCSK9 expression in a sample is measured by detecting a transcribed polynucleotide, or a portion thereof, such as the mRNA of the PCSK9 gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen), or PAX gene (PreAnalytix, Switzerland). Typical assay formats using ribonucleic acid hybridization include nuclear run-on assay, RT-PCR, RNase protection assay (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis.

[0395] In one embodiment, the expression level of PCSK9 is measured using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific PCSK9. Probes can be synthesized by those skilled in the art or obtained from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0396] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe assays. One method for measuring mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PCSK9 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an Affymetrix gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in measuring PCSK9 mRNA levels.

[0397] Alternative methods for measuring the level of expression of PCSK9 in a sample include, for example, RT-PCR (Mullis, 1987, experimental embodiment described in U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, for example, includes the process of nucleic acid amplification (to prepare cDNA) and / or reverse transcriptase of mRNA in a sample, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when they are present in very low numbers. In certain embodiments of the present invention, the level of PCSK9 expression is measured by quantitative fluorescent RT-PCR (i.e., TaqMan™ System).

[0398] The expression level of PCSK9 mRNA can be monitored using membrane blots (such as those used in hybridization analysis such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids).See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference.Measuring PCSK9 expression levels can also include the use of nucleic acid probes in solution.

[0399] In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of these methods is described and illustrated in the Examples provided herein.

[0400] The expression level of PCSK9 protein can be measured using any method known in the art for measuring protein levels, including, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reaction, absorbance spectroscopy, colorimetry, spectrophotometric assay, flow cytometry, (simple or double) immunodiffusion, immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0401] As used herein, the term "sample" includes similar body fluids, cells, or tissues isolated from a subject, as well as collections of body fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluid, etc. Tissue samples can include samples derived from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or body fluids or cells within those organs. In certain embodiments, samples can be derived from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma obtained from a subject. In further embodiments, a "sample derived from a subject" refers to liver tissue obtained from a subject.

[0402] In some embodiments of the method of the present invention, the RNAi agent is administered to a subject so that the RNAi agent is delivered to a specific site in the subject.The inhibition of PCSK9 expression can be evaluated by measuring the level or change in the level of PCSK9 mRNA or PCSK9 protein in a sample derived from body fluid or tissue from a specific site in the subject.In a preferred embodiment, the site is the liver.The site can also be a subsection or subgroup of cells from any one of the above sites.The site can also include cells that express a specific type of receptor.

[0403] V. Methods for treating or preventing diseases associated with PCSK9 The present invention also provides methods for treating or preventing diseases and conditions that can be modulated by downregulating PCSK9 gene expression. For example, the compositions described herein can be used to treat other forms of lipid imbalance, such as hyperlipidemia and conditions associated with these disorders, such as hypercholesterolemia, hypertriglyceridemia, and heart disease and circulatory system disease. Other diseases and conditions that can be modulated by downregulating PCSK9 gene expression include lysosomal storage disorders, including, but not limited to, Niemann-Pick disease, Tay-Sachs disease, lysosomal acid lipase deficiency, and Gaucher disease. The method comprises administering a therapeutically or prophylactically effective amount of an RNAi agent of the present invention to a subject. In one embodiment, the method comprises administering an effective amount of PCSK9 siRNA to a patient with a heterozygous LDLR genotype.

[0404] Preferably, the effect of the decreased PCSK9 gene expression results in a reduction in LDLc (low density lipoprotein cholesterol) levels in the mammal's blood, more particularly serum, hi some embodiments, LDLc levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to pre-treatment levels.

[0405] As used herein, a "subject" includes a human or a non-human animal, preferably a vertebrate, more preferably a mammal. A subject may include a transgenic organism. Most preferably, the subject is a human, such as a human suffering from or susceptible to developing a PCSK9-associated disease.

[0406] In some embodiments of the methods of the invention, PCSK9 expression is reduced for an extended period of time, for example, at least 1 week, 2 weeks, 3 weeks, or 4 weeks or more. For example, in some cases, expression of the PCSK9 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA agent described herein. In some embodiments, the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administration of an iRNA agent. In some embodiments, the PCSK9 gene is suppressed by at least about 85%, 90%, or 95% by administration of a double-stranded oligonucleotide.

[0407] The RNAi agent of the present invention can be administered to a subject by any administration method known in the art, including but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof.In a preferred embodiment, the RNAi agent is administered subcutaneously.

[0408] In some embodiments, administration is by depot injection.Depot injection can continuously release RNAi agent for a long period of time.Therefore, depot injection can reduce the frequency of administration required to obtain desired effect, for example, desired inhibition of PCSK9, or therapeutic or preventive effect.Depot injection can also provide more consistent serum concentration.Depot injection can include subcutaneous injection or intramuscular injection.In a preferred embodiment, depot injection is subcutaneous injection.

[0409] In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, intra-arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver.

[0410] Other administration methods include epidural, intracerebral, intraventricular, intranasal, intraarterial, intracardiac, intraosseous, intrathecal, and intravitreal, and pulmonary. The administration method can be selected based on whether local or systemic treatment is desired and on the site to be treated. The route and site of administration can be selected to facilitate targeting.

[0411] The method includes, for example, administering an iRNA agent at a dose sufficient to reduce the level of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30, or 40 days; and optionally, administering a second, single dose of dsRNA, where the second, single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30, or 40 days after the first, single dose, thereby inhibiting expression of the PCSK9 gene in the subject.

[0412] In one embodiment, a dose of an iRNA agent of the invention is administered no more than once every four weeks, no more than once every three weeks, no more than once every two weeks, or no more than once a week, hi another embodiment, administration can be maintained for one month, two months, three months, or six months, or for one year or more.

[0413] In another embodiment, administration may be provided when low-density lipoprotein cholesterol (LDLc) levels reach or exceed a predetermined minimum level, such as greater than 70 mg / dL, 130 mg / dL, 150 mg / dL, 200 mg / dL, 300 mg / dL, or 400 mg / dL.

[0414] Generally, iRNA agents do not activate the immune system and do not increase cytokine levels, such as, for example, TNF-α or IFN-α levels. For example, when measured by an assay such as an in vitro PBMC assay such as those described herein, the increase in TNF-α or IFN-α levels is less than 30%, 20%, or 10% compared to control cells treated with a control dsRNA, such as a dsRNA that does not target PCSK9.

[0415] For example, a subject may be administered a therapeutic dose of an iRNA agent, such as 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 2.5 mg / kg of dsRNA. The iRNA agent may be administered by intravenous injection over a period of 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes, for example. Administration is repeated periodically, for example, every other week (i.e., every two weeks) for one month, two months, three months, four months, or more. After the initial treatment regimen, the treatment may be administered less frequently. For example, after three months of administration every other week, administration may be repeated once a month for six months, one year, or more. Administration of an iRNA agent can, for example, reduce PCSK9 levels in cells, tissues, blood, urine, or other compartments of a patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0416] Before administering the full dose of the iRNA agent, the patient may be administered a smaller dose (e.g., less than 5% infusion reaction) and monitored for side effects such as allergic reactions, or elevated lipid levels or blood pressure. In another example, the patient may be monitored for undesirable immunostimulatory effects, such as elevated cytokine (e.g., TNF-α or INF-α) levels.

[0417] Efficacy of treatment or prevention is evidenced by a statistically significant improvement in one or more parameters of the disease state, or by the absence of worsening or otherwise expected symptoms. By way of example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50%, or more, in a measurable parameter of the disease may indicate effective treatment. The efficacy of a particular iRNA agent of the invention, or a given formulation of that iRNA agent, can also be determined using an experimental animal model for the given disease, as is known in the art. When using an experimental animal model, efficacy of treatment is demonstrated when a statistically significant reduction in a marker or symptom is observed.

[0418] In one embodiment, the RNAi agent is administered at a dose of about 0.25 mg / kg to about 50 mg / kg, e.g., about 0.25 mg / kg to about 0.5 mg / kg, about 0.25 mg / kg to about 1 mg / kg, about 0.25 mg / kg to about 5 mg / kg, about 0.25 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 5 mg / kg to about 15 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 20 mg / kg to about 30 mg / kg, about 25 mg / kg to about 35 mg / kg, or about 40 mg / kg to about 50 mg / kg.

[0419] In certain embodiments, the RNAi agent is at about 0.25 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 21 mg / kg, about 22 mg / kg, about 23 mg / kg, about 24 mg / kg In one embodiment, the iRNA agent is administered at a dose of about 25 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, 30 mg / kg, about 31 mg / kg, about 32 mg / kg, about 33 mg / kg, about 34 mg / kg, about 35 mg / kg, about 36 mg / kg, about 37 mg / kg, about 38 mg / kg, about 39 mg / kg, about 40 mg / kg, about 41 mg / kg, about 42 mg / kg, about 43 mg / kg, about 44 mg / kg, about 45 mg / kg, about 46 mg / kg, about 47 mg / kg, about 48 mg / kg, about 49 mg / kg, or about 50 mg / kg.

[0420] The dose of an RNAi agent that may be administered to a subject may be adjusted to balance the risks and benefits of a particular dose, for example, to achieve a desired level of PCSK9 gene inhibition (e.g., as assessed based on inhibition of PCSK9 mRNA, expression of PCSK9 protein, or reduction in lipid levels) or a desired therapeutic or prophylactic effect, while avoiding undesirable side effects.

[0421] In some embodiments, the RNAi agent is administered in two or more doses. If it is necessary to facilitate repeated or frequent infusions, implantation of a delivery device, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracapsular, or intraarticular capsule), or a reservoir may be desirable. In some embodiments, the number or amount of subsequent administrations depends on achieving the desired effect, such as suppressing the PCSK9 gene, or achieving a therapeutic or preventive effect, such as reducing the symptoms of hypercholesterolemia. In some embodiments, the RNAi agent is administered according to a schedule. For example, the RNAi agent can be administered once a week, twice a week, three times a week, four times a week, or five times a week. In some embodiments, the schedule includes administration at regular intervals, such as every hour, every four hours, every six hours, every eight hours, every 12 hours, daily, every two days, every three days, every four days, every five days, weekly, every other week, or monthly. In other embodiments, the schedule includes closely spaced administrations followed by a longer period during which the RNAi agent is not administered. For example, the regimen may include an initial set of administrations administered at relatively short intervals (e.g., about every 6 hours, about every 12 hours, about 24 hours, about every 48 hours, or about every 72 hours), followed by a longer period during which the RNAi agent is not administered (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks). In one embodiment, the RNAi agent is initially administered hourly, and then at longer intervals (e.g., daily, weekly, biweekly, or monthly). In another embodiment, the RNAi agent is initially administered daily, and then at longer intervals (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer intervals increase over time or are determined based on achieving the desired effect. In certain embodiments, the RNAi agent is administered once daily for the first week, and then weekly starting on the eighth day of administration. In another specific embodiment, the RNAi agent is administered every other day for the first week, then once a week starting on the eighth day of administration.

[0422] In one embodiment, the iRNA agent is administered twice weekly. In one embodiment, the iRNA agent is administered at a dose of 1 mg / kg twice weekly. In another embodiment, the iRNA agent is administered at a dose of 2 mg / kg twice weekly.

[0423] In one embodiment, the iRNA agent is administered once every two weeks. In one embodiment, the iRNA agent is administered at a dose of 1 mg / kg once every two weeks. In another embodiment, the iRNA agent is administered at a dose of 2 mg / kg once every two weeks.

[0424] In one embodiment, the iRNA agent is administered once a week. In one embodiment, the iRNA agent is administered at a dose of 0.5 mg / kg once a week. In one embodiment, the iRNA agent is administered at a dose of 1 mg / kg once a week. In another embodiment, the iRNA agent is administered at a dose of 2 mg / kg once a week.

[0425] In certain embodiments, the RNAi agent is administered in a dosing regimen that includes an "initial administration phase" of closely spaced administrations, followed by a possible "maintenance phase" in which the RNAi agent is administered at longer intervals. In one embodiment, the initial administration phase includes five daily administrations of the RNAi agent for the first week. In another embodiment, the maintenance phase includes one or two weekly administrations of the RNAi agent. In a further embodiment, the maintenance phase continues for five weeks. In one embodiment, the initial administration phase includes administration of a 2 mg / kg, 1 mg / kg, or 0.5 mg / kg dose five times per week. In another embodiment, the maintenance phase includes administration of a 2 mg / kg, 1 mg / kg, or 0.5 mg / kg dose once, twice, or three times per week, once every two weeks, once every three weeks, once per month, once every two months, once every three months, once every four months, once every five months, or once every six months.

[0426] Any of these regimens may optionally be repeated one or more times, depending on achieving a desired effect, e.g., suppression of the PCSK9 gene, and / or achieving a therapeutic or prophylactic effect, e.g., lowering serum cholesterol levels or reducing the symptoms of hypercholesterolemia.

[0427] In a further embodiment, the administration of the siRNA is administered in combination with an additional therapeutic agent. The siRNA and additional therapeutic agent may be combined in the same composition, e.g., administered parenterally, or the additional therapeutic agent may be administered as part of a separate composition or by another method described herein.

[0428] Examples of additional therapeutic agents include those known to treat lipid disorders such as hypercholesterolemia, atherosclerosis, or dyslipidemia. For example, the siRNA featured in the present invention may be administered with, for example, an HMG-CoA reductase inhibitor (e.g., statins), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acyl-CoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol regulator, a bile acid regulator, a peroxisome proliferator-activated receptor (PPAR) agonist, a gene therapy-based treatment, a combined vasoprotectant (e.g., AGI-1067 from Atherogenics), a glycoprotein Ilb / IIIa inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921 from Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor.Exemplary HMG-CoA reductase inhibitors include atorvastatin (Lipitor® / Tahor / Sortis / Torvast / Cardyl from Pfizer), pravastatin (Pravachol from Bristol-Myers Squibb, Mevalotin / Sanaprav from Sankyo), simvastatin (Zocor® / Sinvacor from Merck, Denan from Boehringer Ingelheim, Lipovas from Banyu), lovastatin (Mevacor / Mevinacor from Merck, Lovastatina from Bexal, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis's Lescol® / Locol / Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay / GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitavastatin (itavastatin / risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, for example, bezafibrate (e.g., Roche's Befizal® / Cedur® / Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil / Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid / Lipur), and ciprofibrate (Sanofi-Synthelabo's Modalim®).Exemplary bile acid sequestrants include, for example, cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme / Sankyo's WelChol™). Exemplary niacin treatments include, for example, immediate-release formulations such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Sustained-release niacin formulations include, for example, Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agents include, for example, aspirin (e.g., Bayer's Aspirin), clopidogrel (Sanofi-Synthelabo / Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Other aspirin-like compounds useful in combination with dsRNA targeting PCSK9 include, for example, Asacard (Pharmacia's extended-release aspirin) and pamicogrel (Kanebo / Angelini Ricerche / CEPA). Exemplary angiotensin-converting enzyme inhibitors include, for example, ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl-CoA cholesterol acetyltransferase (AC AT) inhibitors include, for example, avasimibe (Pfizer), eflucimibe (BioMsrieux Pierre Fabre / Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito).Exemplary cholesterol absorption inhibitors include, for example, ezetimibe (Merck / Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, for example, Torcetrapib (also known as CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, for example, implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol-regulating agents include, for example, NO-1886 (Otsuka / TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst).

[0429] Exemplary bile acid regulators include, for example, HBS-107 (Hisamitsu / Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferator-activated receptor (PPAR) agonists include, for example, tesaglitazar (AZ-242) (AstraZeneca), netoglitazone (MCC-555) (Mitsubishi / Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals / GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals / GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene-based therapies include, for example, AdGWEGF 121.10 (GenVec), ApoA1 (UCB Pharma / Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics / Incyte, Aventis, Xenon). Exemplary glycoprotein Ilb / IIIa inhibitors include, for example, roxifiban (also known as DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA / Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).Exemplary squalene synthase inhibitors include, for example, BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). Exemplary MCP-I inhibitors include, for example, RS-504393 (Roche Bioscience). Anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals) and nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also suitable for administration in combination with the dsRNA featured in the present invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, for example, advicor (niacin / lovastatin from Kos Pharmaceuticals), amlodipine / atorvastatin (Pfizer), and ezetimibe / simvastatin (e.g., Vytorin® 10 / 10, 10 / 20, 10 / 40, and 10 / 80 tablets from Merck / Schering-Plough Pharmaceuticals).Agents for treating hypercholesterolemia that are suitable for administration in combination with a dsRNA targeting PCSK9 include, for example, lovastatin, niacin Altoprev® extended-release tablets (Andrx Labs), lovastatin Caduet® tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® tablets (AstraZeneca), rosuvastatin calcium Lescol® capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® tablets (Parke-Davis), atorvastatin calcium Lofibra® capsules (Gate), Niaspan extended-release tablets (Kos), niacin Pravachol tablets (Bristol-Myers Squibb). Squibb), pravastatin sodium TriCor® tablets (Abbott), fenofibrate Vytorin® 10 / 10 tablets (Merck / Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ tablets (Sankyo), colesevelam hydrochloride Zetia® tablets (Schering), ezetimibe Zetia® tablets (Merck / Schering-Plough Pharmaceuticals), and ezetimibe Zocor® tablets (Merck).

[0430] In one embodiment, the iRNA agent is administered in combination with an ezetimibe / simvastatin combination (e.g., Vytorin® (Merck / Schering-Plough Pharmaceuticals)). In one embodiment, the iRNA agent is administered to a patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered simultaneously.

[0431] In another aspect, the invention features a method of instructing an end user, e.g., a caregiver or subject, on how to administer an iRNA agent described herein. The method optionally includes providing one or more doses of the iRNA agent to the end user and instructing the end user to administer the iRNA agent in a dosing regimen described herein, thereby instructing the end user.

[0432] In one embodiment, the present invention provides a method of treating a patient by selecting the patient based on the patient's need for LDL reduction, LDL reduction without HDL reduction, ApoB reduction, or total cholesterol reduction, comprising administering to the patient an siRNA in an amount sufficient to reduce the patient's LDL or ApoB levels, for example, without substantially reducing HDL levels.

[0433] Genetic predisposition plays a role in the development of diseases related to target genes, such as hyperlipidemia.Therefore, by taking family history or by screening one or more genetic markers or genetic mutations, patients who need siRNA can be identified.Examples of genes involved in hyperlipidemia include, but are not limited to, LDL receptor (LDLR), apolipoprotein (ApoA1, ApoB, ApoE, etc.), cholesteryl ester transfer protein (CETP), lipoprotein lipase (LPL), hepatic lipase (LIPC), endothelial lipase (EL), and lecithin cholesterol acyltransferase (LCAT).

[0434] A medical care provider, such as a doctor, nurse, or family member, can ask about family history before prescribing or administering the iRNA agent of the present invention.Furthermore, tests can be performed to determine genotype or phenotype.For example, before PCSK9 dsRNA is administered to a patient, DNA testing can be performed on a sample, such as a blood sample, from the patient to identify the PCSK9 genotype and / or phenotype.In another embodiment, tests are performed to identify related genotypes and / or phenotypes, such as LDLR genotypes. Examples of genetic mutations related to the LDLR gene can be found in the art, for example, in the following publications, which are incorporated by reference: Costanza et al. (2005) Am J Epidemiol. 15; 161(8): 714-24; Yamada et al. (2008) J Med Genet. Jan; 45(1): 22-8, Epub 2007 Aug 31; and Boes et al. (2009) Exp. Gerontol 44: 136-160, Epub 2008 Nov 17.

[0435] VI. Kit The present invention also provides kits for using any of the iRNA agents and / or performing any of the methods of the present invention. Such kits include one or more RNAi agents and instructions for use, e.g., for inhibiting expression of PCSK9 in a cell by contacting the cell with an amount of the RNAi agent effective to inhibit expression of PCSK9. The kits may optionally further include a means for contacting the cell with the RNAi agent (e.g., an injection device) or a means for measuring inhibition of PCSK9 (e.g., a means for measuring inhibition of PCSK9 mRNA or TTR protein). Such a means for measuring inhibition of PCSK9 may include a means for obtaining a sample from a subject, such as, for example, a plasma sample. The kits of the present invention may optionally further include a means for administering the RNAi agent to a subject or a means for determining a therapeutically or prophylactically effective amount.

[0436] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary 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 characterizing the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. [Example]

[0437] Materials and Methods The following materials and methods were used in the examples.

[0438] cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat #4368813) A master mix of 2 μl of 10x buffer, 0.8 μl of 25x dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of HO was added to 10 μl of total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermocycler (Hercules, CA) through the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and a 4°C hold.

[0439] Cell culture and transfection Hep3B, HepG2, or HeLa cells (ATCC, Manassas, VA) were grown to near confluence in the recommended medium (ATCC) supplemented with 10% FBS and glutamine (ATCC) at 37°C in a 5% CO atmosphere and then released from the plate by trypsinization. For duplexes screened in a 96-well format, transfection was performed by adding 44.75 μl of Opti-MEM and 0.25 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cat # 13778-150) per well to 5 μl of each siRNA duplex in individual wells within a 96-well plate. The mixture was then incubated at room temperature for 15 minutes. Approximately 2 × 10 4 50 μl of complete growth medium without antibiotics containing cells was added to the siRNA mixture. For duplexes screened in a 384-well format, 5 μl of Opti-MEM and 0.1 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA. cat # 13778-150) were mixed with 5 μl of each siRNA duplex per individual well. The mixture was then incubated at room temperature for 15 minutes, after which approximately 8 × 10 cells were cultured. 3 An additional 40 μl of complete growth medium without antibiotics containing the cells was added. Cells were incubated for 24 hours before RNA purification. Single dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose response experiments were performed using 8 x 5-fold serial dilutions starting at 2 nM.

[0440] Free uptake transfection 5 μl of each GalNac-conjugated siRNA in PBS was added to each well of a 96-well plate to 3 × 10 siRNAs resuspended in 95 μl of In Vitro Gro CP medium (In Vitro Technologies-Celsis, Baltimore, MD). 4freshly thawed cryopreserved cynomolgus monkey hepatocytes (In Vitro Technologies-Celsis, Baltimore, MD; lot#JQD), or for a 384-well plate format, 5 ul of siRNA and 1.2 x 10 3 The siRNA was combined with 45 μl of medium containing 1000 cells. The mixture was incubated at 37°C in a 5% CO atmosphere for approximately 24 hours. The siRNA was tested at multiple concentrations, ranging from 500 to 0.1 nM, in single-dose experiments and using 8 x 5-fold serial dilutions starting at 500 nM in dose-response experiments.

[0441] Total RNA isolation using DYNABEADS mRNA isolation kit (Invitrogen, part #:610-12) Cells were harvested and lysed in 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl of lysis / binding buffer mixture were added to a round-bottom plate and mixed for 1 minute. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were captured again, and the supernatant was removed. Next, the beads were washed with 150 μl of wash buffer B, captured, and the supernatant was removed. Next, the beads were washed with 150 μl of elution buffer, captured, and the supernatant was removed. The beads were dried for 2 minutes. After drying, 50 μl of elution buffer was added and mixed at 70°C for 5 minutes. The beads were captured on a magnet for 5 minutes, and 50 μl of the supernatant was removed and added to another 96-well plate.

[0442] For the 384-well format, cells were lysed for 1 minute, followed by the addition of 50 μl of lysis / binding buffer. 2 μl of magnetic beads were used per well. The required volume of beads was aliquoted, captured on a magnetic stand, and the bead storage solution was removed. The beads were then resuspended in the required volume of lysis / binding buffer (25 μl per well), and 25 μl of the bead suspension was added to the lysed cells. The lysate-bead mixture was incubated for 10 minutes in a VibraTransaltor (Union Scientific Corp., Randallstown, MD) at setting number 7. The beads were then captured using a magnetic stand, the supernatant was removed, and the beads were washed once with 90 μl of buffer A, followed by one wash step with 90 μl of buffer B and 100 μl of elution buffer. The beads were soaked in each wash buffer for approximately 1 minute (without mixing). After the final washing step, the beads were resuspended in 15 μl of elution buffer for 5 min at 70°C, after which the beads were captured and the supernatant (maximum 8 μl) was removed for cDNA synthesis and / or storage of purified RNA (−20°C).

[0443] Real-time PCR Two microliters of cDNA was mixed per well in a 384-well plate (Roche cat # 04887301001) with 0.5 μl of human GAPDH TaqMan probe (Applied Biosystems Cat #4326317E) for human cells, 0.5 μl of human PCSK9 TaqMan probe (Applied Biosystems cat # Hs03037355_m1), or 0.5 μl of cynomolgus monkey GAPDH custom TaqMan Assay (150 nM cyno GAP F primer - 5'GCATCCTGGGCTACACTGA (SEQ ID NO: 5); 150 nM cyno GAP R primer - 5'-TGGGTGTCGCTGTTGAAGTC (SEQ ID NO: 6) 250 nM cyno GAP The resulting mixture was added to a master mix containing 0.5 μl of a cyno PCSK9 custom TaqMan Assay (900 nM cyno PCSK9 F primer 5′-ACGTGGCTGGCATTGCA (SEQ ID NO: 8); 900 nM cyno PCSK9 R primer 5′-AAGTGGATCAGTCTCTGCCTCAA (SEQ ID NO: 9); 250 nM cyno PCSK9 probe 5′-6FAM-CATGATGCTGTCTGCCGAGCCG-BHQ1-Q-3′ (SEQ ID NO: 10)), and 5 μl of Lightcycler 480 Probe Master Mix (Roche Cat #04887301001). Real-time PCR was performed in a Roche LC480 Real Time PCR System (Roche) using the ΔΔCt(RQ) assay. Unless otherwise stated, each duplex was tested in two independent transfections, and each transfection was assayed in duplicate.

[0444] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. For free uptake assays, data were normalized to cells treated with PBS or GalNAc-1955 (the highest concentration used for experimental compounds). IC was calculated using a four-parameter fitting model using XLFit. 50 was calculated and normalized to cells transfected with AD-1955 over the same dose range or to the lowest dose by itself.

[0445] The sense and antisense sequences of AD-1955 are sense: 5'-cuuAcGcuGAGuAcuucGAdTsdT-3' (SEQ ID NO: 11); and antisense: 5'-UCGAAGuACUcAGCGuAAGdTsdT-3' (SEQ ID NO: 12).

[0446] [Table 3]

[0447] [Table 4]

[0448] [Table 5]

[0449] Example 1. Synthesis of GalNAc-conjugated oligonucleotides Using the above-described technique, a series of siRNA duplexes spanning the sequence of PCSK9 mRNA are designed and synthesized, and are conjugated with trivalent GalNAc at the 3' end of the sense strand.The sequences of these duplexes are shown in Table 1.These same sequences with various nucleotide modifications are also synthesized, and are conjugated with trivalent GalNAc.The sequences of the modified duplexes are shown in Table 2.

[0450]

Table 6

[0451]

Table 7

[0452]

Table 8

[0453]

Table 9

[0454]

Table 10

[0455]

Table 11

[0456]

Table 12

[0457]

Table 13

[0458]

Table 14

[0459]

Table 15

[0460] Table 16

[0461] Table 17

[0462] Table 18

[0463] Table 19

[0464] Table 20

[0465] Table 21

[0466] Table 22

[0467] Table 23

[0468] Table 24

[0469] Table 25

[0470] Table 26

[0471] Table 27

[0472] Table 28

[0473] Table 29

[0474]

Table 30

[0475] Table 31

[0476] Table 32

[0477] Table 33

[0478] Table 34

[0479] Table 35

[0480] Table 36

[0481] Table 37

[0482] Table 38

[0483] Table 39

[0484] Table 40

[0485] Table 41

[0486] Table 42

[0487] Table 43

[0488] Table 44

[0489] Table 45

[0490] Table 46

[0491] Table 47

[0492] Table 48

[0493] Table 49

[0494]

Table 50

[0495] Table 51

[0496] Table 52

[0497] Table 53

[0498] Table 54

[0499] Table 55

[0500] Table 56

[0501] Table 57

[0502] [Table 58]

[0503] [Table 59]

[0504] [Table 60]

[0505] [Table 61]

[0506] [Table 62]

[0507] Example 2. In vitro and in vivo screening A subset of these duplexes was evaluated for efficacy in single-dose free uptake assays in cynomolgus monkey hepatocytes. Briefly, primary cynomolgus monkey hepatocytes (PCH) were treated with conjugated modified siRNA duplexes at three concentrations: 500nM, 100nM, and 10nM. The free uptake assays of 100nM and 10nM were performed twice, and data are expressed as the mean message remaining relative to the control ± standard deviation (SD). The 500nM screening was performed once. Table 3 shows the results of these assays.

[0508] [Table 63]

[0509] [Table 64]

[0510] [Table 65]

[0511] [Table 66]

[0512] [Table 67]

[0513] [Table 68]

[0514] The modified, conjugated PCSK9 siRNA duplexes were also evaluated for efficacy by transfection assays in three human cell lines. PCSK9 siRNA was transfected into three different cell lines, HeLa, Hep3B, and HepG2, at two doses: 10 nM and 0.1 nM. The results of these assays are shown in Table 4, where the data are expressed as the percentage of message remaining relative to the control.

[0515] Figure 1 shows that there is a general reproducibility of the silencing activity of PCSK9 duplexes between free uptake and transfection assays.

[0516] IC of selected duplexes by free uptake in cynomolgus cells and transfection in Hep3B cells 50 The values ​​are shown in Table 5.

[0517] [Table 69]

[0518] [Table 70]

[0519] [Table 71]

[0520] [Table 72]

[0521] [Table 73]

[0522] [Table 74]

[0523] [Table 75]

[0524] AD-48400 was also assayed for in vivo efficacy in female mice carrying a human PCSK9 transgene randomly inserted into their genome without disrupting the endogenous PCSK9 gene. Briefly, mice were subcutaneously injected with a single 20 mg / kg dose on day 0, a single 100 mg / kg dose on day 0, and five 20 mg / kg doses on days 0, 1, 2, 3, 4, and 5. Serum was collected on days -6, -3, 0, 1, 2, 3, 4, and 7, and PCSK9 protein levels were measured by ELISA assay. The results of these analyses are shown in Figure 2 and demonstrate a dose-response effect of AD-48400 conjugated to GalNAc at all three doses tested.

[0525] The six most effective duplexes identified by the in vitro screening described above were evaluated for in vivo efficacy and duration of response. Transgenic PCSK9 mice were injected with 5 mg / kg or 25 mg / kg of either AD-48400, AD-53830, AD-53806, AD-53815, AD-53748, or AD-53798 on days 0, 1, 2, 3, and 4. Serum PCSK9 protein levels were measured by ELISA on days -3, 0, 1, 2, 3, 4, 8, 11, 15, 18, 22, 26, 31, and 36. The results are shown in Figures 3A and 3B.

[0526] Example 3. Lead optimization Based on the efficacy assay described in Example 2 above, PCSK9 siRNAs based on the parental sequences of AD-53815 and AD-53806 with various chemical modifications were evaluated for efficacy in a free uptake assay in primary cynomolgus monkey hepatocytes (PCH) at 200 nM, 20 nM, 2 nM, and 0.2 nM. All doses except the 0.2 nM dose were assayed twice, and data are expressed as the average fraction of message remaining relative to the control. The 0.2 nM dose was assayed once. The results of these assays are shown in Table 6.

[0527] [Table 76]

[0528] [Table 77]

[0529] [Table 78]

[0530] [Table 79]

[0531] [Table 80]

[0532] [Table 81]

[0533] siRNAs with various chemical modifications based on the parent sequences of AD-53815 and AD-53806 were also screened for in vitro efficacy by transfection in Hep3B cells at 10 nM and 0.1 nM. The results of this structure-activity relationship screen are shown in Table 7 and are expressed as the mean percentage of message remaining relative to the control ± SD.

[0534] [Table 82]

[0535] [Table 83]

[0536] [Table 84]

[0537] [Table 85]

[0538] [Table 86]

[0539] [Table 87]

[0540] To determine whether any of the siRNAs from the in vitro SAR screen were more effective at silencing PCSK9 than the parent siRNA (AD-53815), PCSK9 transgenic mice were administered a single 3 mg / kg dose of the siRNAs shown in Figure 4, and 72 hours after administration, PCSK9 protein levels were measured by ELISA assay. The results, shown in Figure 5, demonstrate that AD-57928 is surprisingly effective at silencing PCSK9. Figure 6 demonstrates that not only does a single administration of AD-57928 effectively knockdown PCSK9 protein, but there is also a dose-response with AD-57928.

[0541] Example 4. Split-dose test using AD-57928 The ability of AD-57928 to suppress PCSK9 protein expression was evaluated by measuring the levels of human PCSK9 (hPCSK9) protein in the serum of hPCSK9 transgenic mice after administration of AD-57928. As described in Table 8 below, AD-57928 was administered subcutaneously using six different dosing regimens, including an "initial dosing phase" (0.5 mg / kg, 1 mg / kg, or 2 mg / kg once daily for five consecutive days) during the first week, followed by a "maintenance phase" (0.5 mg / kg, 1 mg / kg, or 2 mg / kg once or twice weekly for five weeks). The final dose was administered on day 38. Each dosing regimen was tested using groups of three mice, including two males and one female. A control group received an injection of PBS.

[0542] [Table 88]

[0543] Serum was collected 3 days before the first dose and 1, 4, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 42, 45, 52, 59, and 65 days after the first dose. PCSK9 protein levels in serum were assessed by ELISA assay. The results are shown in Figures 6, 7, and 8.

[0544] Reductions in hPCSK9 serum protein levels were observed 72 hours after the first dose and persisted for up to 38 days. Administration of AD-57928 at initial doses of 5×2 mg / kg, 5×1 mg / kg, and 5×0.5 mg / kg resulted in approximately 90%, 70%, and 60% reductions in hPCSK9 serum protein levels, respectively (see Figures 6-8). In the group administered using the 2× maintenance regimen, reduced levels of hPCSK9 persisted for one week longer than in the group administered using the 1× maintenance regimen, returning to baseline 4 weeks after the final dose (see Figures 6-8).

[0545] Example 5. Phosphorothioate titration To determine the effect of the number and position of phosphorothioate modifications on the ability of dsRNA to inhibit PCSK9 expression, several siRNAs based on the parent sequences of AD-57928, AD-53806, and AD-53830 were prepared and tested, as shown in Table 9. To determine whether any of the siRNAs were more effective at silencing PCSK9 than AD-57928, PCSK9 transgenic mice were administered a single 0.3 mg / kg dose of the siRNAs in Table 9, and 72 hours after administration, PCSK9 protein levels were measured by ELISA assay. The results, shown in Figure 9, surprisingly demonstrate that AD-57928 is effective at silencing PCSK9. AD-58893, AD-58894, AD-58896, AD-58897, AD-58898, and AD-58899 were also able to silence PCSK9 compared to the control.

[0546] [Table 89]

[0547] Example 6. Liver drug levels of AD-57928 and AD-58895 The purpose of this study was to quantify siRNA levels in the livers of wild-type mice to define appropriate conditions for drug level screening. The siRNAs used in the experiment were AD-57928 and AD-58895 (which did not reduce PCSK9 protein levels in Example 5). AD-58895 was used as a comparative example to define a time point at which differences in dr...

Claims

1. 1. A double-stranded RNAi agent capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; Each n may or may not be present ｐ , n ｐ ', n ｑ , and n ｑ ' independently represent an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; N ｂ the modification on Y is different from the modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

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

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

4. 2. The double-stranded RNAi agent of claim 1, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

5. 2. The double-stranded RNAi agent of claim 1, wherein the YYY motif is present at or near the cleavage site of the sense strand.

6. 2. The double-stranded RNAi agent of claim 1, wherein the Y'Y'Y' motif is present at positions 11, 12, and 13 of the antisense strand at the 5' end.

7. The double-stranded RNAi agent of claim 6, wherein Y' is 2'-O-methyl.

8. Formula (III) is represented by formula (IIIa): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -Y'Y'Y'-N ａ’ -n ｑ’ 5'(IIIa) The double-stranded RNAi agent of claim 1 , represented by:

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

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

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

12. 2. The double-stranded RNAi agent of claim 1, wherein the double-stranded region is 15 to 30 nucleotide pairs in length.

13. 13. The double-stranded RNAi agent of claim 12, wherein the double-stranded region is 17 to 23 nucleotide pairs in length.

14. 13. The double-stranded RNAi agent of claim 12, wherein the double-stranded region is 17 to 25 nucleotide pairs in length.

15. 13. The double-stranded RNAi agent of claim 12, wherein the double-stranded region is 23 to 27 nucleotide pairs in length.

16. 13. The double-stranded RNAi agent of claim 12, wherein the double-stranded region is 19 to 21 nucleotide pairs in length.

17. 13. The double-stranded RNAi agent of claim 12, wherein the double-stranded region is 21 to 23 nucleotide pairs in length.

18. 10. The double-stranded RNAi agent of claim 1, wherein each strand has 15 to 30 nucleotides.

19. 2. The double-stranded RNAi agent of claim 1, wherein the modification on the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

20. 20. The double-stranded RNAi agent of claim 19, wherein the modification on the nucleotide is a 2'-O-methyl or a 2'-fluoro modification.

21. 2. The double-stranded RNAi agent of claim 1, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

22. The ligand is 【Chemical 1】 The double-stranded RNAi agent of claim 1, wherein

23. The double-stranded RNAi agent of claim 1 , wherein the ligand is attached to the 3′ end of the sense strand.

24. Schematic diagram below 【Chemistry 2】 24. The double-stranded RNAi agent of claim 23, conjugated to the ligand of the formula: wherein X is O or S.

25. 10. The double-stranded RNAi agent of claim 1, further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

26. 26. The double-stranded RNAi agent of claim 25, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand.

27. 27. The double-stranded RNAi agent of Claim 26, wherein the strand is the antisense strand.

28. 27. The double-stranded RNAi agent of Claim 26, wherein the strand is the sense strand.

29. 26. The double-stranded RNAi agent of claim 25, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand.

30. 30. The double-stranded RNAi agent of Claim 29, wherein said strand is the antisense strand.

31. 30. The double-stranded RNAi agent of Claim 29, wherein said strand is the sense strand.

32. 26. The double-stranded RNAi agent of claim 25, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand.

33. 33. The double-stranded RNAi agent of Claim 32, wherein said strand is the antisense strand.

34. 2. The double-stranded RNAi agent of claim 1, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

35. 2. The double-stranded RNAi agent of claim 1, wherein the Y nucleotide comprises a 2'-fluoro modification.

36. 2. The double-stranded RNAi agent of claim 1, wherein the Y' nucleotide comprises a 2'-O-methyl modification.

37. The double-stranded RNAi agent of claim 1, wherein p'>0.

38. The double-stranded RNAi agent of claim 1, wherein p'=2.

39. 39. The double-stranded RNAi agent of claim 38, wherein q'=0, p=0, q=0, and p' overhanging nucleotides are complementary to the target mRNA.

40. 39. The double-stranded RNAi agent of claim 38, wherein q'=0, p=0, q=0, and the p' overhanging nucleotides are non-complementary to the target mRNA.

41. 30. The double-stranded RNAi agent of claim 28, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

42. At least one n ｐ 42. The double-stranded RNAi agent of any one of claims 37-41, wherein ' is linked to an adjacent nucleotide via a phosphorothioate bond.

43. All n ｐ 43. The double-stranded RNAi agent of claim 42, wherein ' is linked to an adjacent nucleotide via a phosphorothioate linkage.

44. 2. The double-stranded RNAi agent of claim 1, selected from the group of RNAi agents listed in Table 1, Table 2, Table 9, Table 10, Table 12, and FIG. 12A.

45. 2. The double-stranded RNAi agent of claim 1, selected from the group consisting of AD-53815, AD-56663, AD-56658, AD-56676, AD-56666, AD-57928, and AD-60212.

46. 1. A double-stranded RNAi agent capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; Each n may or may not be present ｐ , n ｐ ', n ｑ , and n ｑ ' independently represent an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from the modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

47. 1. A double-stranded RNAi agent capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present ｐ , n ｑ , and n ｑ ' independently represent an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is attached to the adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from the modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

48. 1. A double-stranded RNAi agent capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present ｐ , n ｑ , and n ｑ ' independently represent an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is attached to the adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from the modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. A double-stranded RNAi agent represented by:

49. 1. A double-stranded RNAi agent capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may or may not be present ｐ , n ｑ , and n ｑ ' independently represent an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is attached to the adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from the modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand comprises at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. A double-stranded RNAi agent represented by:

50. 1. A double-stranded RNAi agent capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding PCSK9, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-Y'Y'Y'-N ａ '-n ｑ '5' (IIIa) (In the formula: Each n may or may not be present ｐ , n ｑ , and n ｑ ' independently represent an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is attached to the adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; the sense strand comprises at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. A double-stranded RNAi agent represented by:

51. An RNAi agent selected from the group of RNAi agents listed in Table 1, Table 2, Table 9, Table 10, Table 12, and Figure 12A.

52. 52. A cell containing the double-stranded RNAi agent of any one of claims 1 and 46-51.

53. 52. A pharmaceutical composition comprising the double-stranded RNAi agent of any one of claims 1 and 46-51.

54. 54. The pharmaceutical composition of claim 53, wherein the RNAi agent is administered in a non-buffered solution.

55. 55. The pharmaceutical composition of claim 54, wherein the non-buffered solution is saline or water.

56. 54. The pharmaceutical composition of claim 53, wherein the siRNA is administered with a buffer.

57. 57. The pharmaceutical composition of claim 56, wherein the buffer comprises an acetate buffer, a citrate buffer, a prolamine buffer, a carbonate buffer, or a phosphate buffer, or any combination thereof.

58. 58. The pharmaceutical composition of claim 57, wherein the buffer is phosphate buffered saline (PBS).

59. 1. A method for inhibiting PCSK9 expression in a cell, comprising: (a) contacting the cell with the double-stranded RNAi agent of any one of claims 1 and 46-51 or the pharmaceutical composition of any one of claims 53-58; (b) maintaining the cell produced in step (a) for a sufficient time to obtain the degradation of the mRNA transcript of PCSK9 gene, thereby inhibiting the expression of PCSK9 gene in said cell.

60. 60. The method of claim 59, wherein the cell is in a subject.

61. 61. The method of claim 60, wherein the subject is a human.

62. 62. The method of any one of claims 59 to 61, wherein the PCSK9 expression is inhibited by at least about 30%.

63. 57. A method of treating a subject suffering from a disorder mediated by PCSK9 expression, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 46-51 or the pharmaceutical composition of any one of claims 53-58, thereby treating the subject.

64. 64. The method of claim 63, wherein the subject is a human.

65. 65. The method of claim 64, wherein the human is suffering from hypercholesterolemia.

66. 64. The method of claim 63, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

67. 67. The method of claim 66, wherein the double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg.

68. 67. The method of claim 66, wherein the double-stranded RNAi agent is administered subcutaneously.

69. 67. The method of Claim 66, wherein the double-stranded RNAi agent is administered intravenously.

70. 67. The method of claim 66, wherein the RNAi agent is administered in two or more doses.

71. the RNAi agent is administered in a dosing regimen comprising an initial administration phase followed by a maintenance phase; the initial administration step comprising administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg five times per week; 71. The method of claim 70, wherein the maintenance phase comprises administering a dose of 2 mg / kg, 1 mg / kg or 0.5 mg / kg once per week, twice per week, three times per week, once every two weeks, once per three weeks, once per month, once per two months, once per three months, once per four months, once per five months, or once per six months.

72. 71. The method of claim 70, wherein the RNAi agent is administered at an interval selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

73. 57. A method of treating hypercholesterolemia in a subject, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of any one of claims 1 and 46-51 or the pharmaceutical composition of any one of claims 53-58, thereby treating the subject.

74. 74. The method of claim 73, wherein the subject is a primate or a rodent.

75. 74. The method of claim 73, wherein the subject is a human.

76. 74. The method of claim 73, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

77. 74. The method of claim 73, wherein the double-stranded RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg.

78. 74. The method of Claim 73, wherein the RNAi agent is administered in two or more doses.

79. the RNAi agent is administered in a dosing regimen comprising an initial administration phase followed by a maintenance phase; the initial administration step comprising administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg five times per week; 74. The method of claim 73, wherein the maintenance phase comprises administering a dose of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg once, twice, or three times weekly, once every two weeks, once every three weeks, once every month, once every two months, once every three months, once every four months, once every five months, or once every six months.

80. 74. The method of Claim 73, wherein the RNAi agent is administered at an interval selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

81. 74. The method of Claim 73, wherein the double-stranded RNAi agent is administered subcutaneously.

82. 74. The method of Claim 73, wherein the double-stranded RNAi agent is administered intravenously.

83. 74. The method of claim 73, further comprising determining the subject's LDLR genotype or phenotype.

84. 74. The method of claim 73, wherein administration lowers serum cholesterol in the subject.

85. 74. The method of claim 73, further comprising determining the serum cholesterol level in the subject.

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