PCSK9 iRNA Compositions and Methods of Use Thereof

Abstract

To provide methods of using RNAi agents and methods of treating subjects having lipid disorders such as hyperlipidemia.SOLUTION: The invention relates to RNAi agents, e.g., double-stranded RNAi agents, targeting the PCSK9 gene, and use of such RNAi agents to inhibit expression of PCSK9.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 hereby incorporated by reference in its entirety. The ASCII copy, created on October 29, 2013, is named 121301-00420_SL.txt and is 433,512 bytes in size. [Background technology]

[0003] Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family. Eight other 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 a role 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 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). PCSK9 may also play a role in determining LDL cholesterol levels in the general population, as single nucleotide polymorphisms (SNPs) are associated with cholesterol levels in the Japanese population (Non-Patent Document 12).

[0005] Autosomal dominant hypercholesterolemia (ADH) is a single gene disease in which patients show elevated total and LDL cholesterol levels, tendon xanthomas, and early atherosclerosis (Non-Patent Document 13). The pathogenesis 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 the endocytosis of the LDLR-LDL complex through its interaction with clathrin. Therefore, if PCSK9 mutations are responsible in the Hchola3 family, it is likely that PCSK9 plays a role in receptor-mediated LDL uptake.

[0006] Overexpression studies point to the role of PCSK9 in controlling LDLR levels and thus 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 for 3 or 4 days in mice leads to increased 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 leads to a significant decrease in hepatic LDLR protein without affecting LDLR mRNA levels, SREBP protein levels, or the SREBP protein nuclear to cytoplasmic ratio.

[0007] Although hypercholesterolemia itself is asymptomatic, long-term elevation of serum cholesterol can lead to atherosclerosis. Over decades, chronically elevated serum cholesterol can cause the formation of atheromatous plaques in arteries, which can lead to progressive narrowing or even complete blockage of affected arteries. Furthermore, smaller plaques can rupture and form blood clots, impeding blood flow, leading to, for example, myocardial infarction and / or stroke. If the formation of narrowing or blockage is gradual, blood supply to tissues and organs is slowly reduced until organ function is compromised. [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

Non-licensed Document 5

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

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Non-licensed Document 17

[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, such as double-stranded iRNA agents, that target PCSK9 are disclosed herein.Methods of using the compositions of the present invention for inhibiting PCSK9 expression and treating pathologies associated with PCSK9 expression, such as hypercholesterolemia, are also disclosed.

[0011] Accordingly, in one 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; 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 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 site of cleavage in the sense strand.

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

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

[0017] In one embodiment, 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) It is represented by:

[0018] In another embodiment, formula (III) is represented by 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) It is represented 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) It is represented by:

[0020] In one embodiment, formula (III) is represented by 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, a and N a ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides. It is represented by:

[0021] In one embodiment, the double-stranded region is 15-30 nucleotide pairs long. In another embodiment, the double-stranded region is 17-23 nucleotide pairs long. In yet another embodiment, the double-stranded region is 17-25 nucleotide pairs long. In one embodiment, the double-stranded region is 23-27 nucleotide pairs long. In another embodiment, the double-stranded region is 19-21 nucleotide pairs long. In another embodiment, the double-stranded region is 21-23 nucleotide pairs long. In one embodiment, each strand has 15-30 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] It 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, hi 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 linked to the adjacent nucleotide via a phosphorothioate bond.

[0037] In one embodiment, all n p ' is linked 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 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 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 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, the 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 ' 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 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 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, the 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 ' 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 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, the 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 ' 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 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'-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 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, the 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 ' 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 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 -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 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, the modifications being 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 invention also provides cells, vectors, host cells, and pharmaceutical compositions comprising the double-stranded RNAi agents of the 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, prolamine 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 includes contacting a cell with an RNAi agent, such as a double-stranded RNAi agent or vector of the present invention; and maintaining the cell generated in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the PCSK9 gene, thereby inhibiting the 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 comprises administering a therapeutically effective amount of an RNAi agent, such as 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 suffers from 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, the loading phase including administering a dose of 2 mg / kg, 1 mg / kg or 0.5 mg / kg five times per week, and the maintenance phase including 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 to the subject a therapeutically effective amount of an RNAi agent, e.g., a double-stranded RNAi agent, or vector, of the invention, 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, the initial dosing phase including administering a dose of 2 mg / kg, 1 mg / kg or 0.5 mg / kg five times per week, and the maintenance phase including 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 description 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. [Diagram 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. [Diagram 5] 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] FIG. 1 is a graph showing levels of PCSK9 protein in serum of PCSK9 transgenic mice following administration of AD-57928 at a dose of 5×2 mg / kg during the “initiation phase” and at doses of 1×2 mg / kg or 2×2 mg / kg during the “maintenance phase.” [Figure 7] FIG. 1 is a graph showing levels of PCSK9 protein in serum of PCSK9 transgenic mice following administration of AD-57928 at a dose of 5×1 mg / kg during the "initiation phase" and at doses of 1×1 mg / kg or 2×1 mg / kg during the "maintenance phase." [Figure 8] FIG. 1 is a graph showing levels of PCSK9 protein in serum of PCSK9 transgenic mice following administration of AD-57928 at a dose of 5×0.5 mg / kg during the “initiation 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 following 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 theoretical amount in the liver of C57B6 wild type mice following 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 following 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 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 qdx5+qwx3. Figure 14B is a graph showing the absolute amount of PCSK9 protein in serum of non-human primates after administration of the indicated iRNA agent qdx5+qwx3. [Figure 15] 1 is a graph showing the amount of low density lipoprotein cholesterol (LDL or LDLc) in serum of non-human primates, expressed as a percentage of LDL remaining relative to pre-bleed levels of LDL, following administration of the indicated iRNA agent at qdx5+qwx3. [Figure 16]Figure 16A is a graph showing the amount of low density lipoprotein cholesterol (LDL or LDLc) in serum of non-human primates expressed as a percentage of the average 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 the amount of pre-bleed in 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 serum of non-human primates expressed as a percentage of the average 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 administration of the 2 mg / kg, 2xw group was on day 36. Figure 17B is a graph showing the amount of PCSK9 protein versus pre-bleed amount in 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 serum of non-human primates, expressed as a percentage of LDL remaining relative to pre-bleed levels of LDL, following administration of the indicated iRNA agent at qdx5+qwx3. [Figure 19] 1 is a graph showing the amount of low density lipoprotein cholesterol (LDL or LDLc) in serum of non-human primates, expressed as a percentage of LDL remaining relative to pre-bleed levels of LDL, following administration of the indicated iRNA agent at qdx5+qwx3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 for inhibiting PCSK9 expression and treating pathologies associated with PCSK9 expression, e.g., hypercholesterolemia, are also disclosed.

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

[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 (whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. GI:299523249); mouse PCSK9 (whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. GI:163644257); and rat PCSK9 (whose amino acid and nucleotide sequences 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 the mRNA that is the product of RNA processing of the 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 shown using standard nucleotide nomenclature.

[0080] "G", "C", "A" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine and uracil as a base, respectively. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide in which the nucleobase is thymine, e.g., deoxyribothymine, 2'-deoxythymidine or thymidine. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to modified nucleotides, or surrogate replacement moieties, as further detailed below. Those skilled in the art will appreciate that guanine, cytosine, adenine and uracil can be replaced by other moieties without appreciably altering the base pairing properties of an oligonucleotide containing a nucleotide having such a replacement moiety. 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 replaced in the nucleotide sequences of the invention by nucleotides containing, for example, inosine, and sequences containing such replacement moieties are embodiments of the invention.

[0081] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent", as used interchangeably herein, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as the term is defined herein. iRNA directs sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) the expression of PCSK9 in a cell, e.g., a cell 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, e.g., a PCSK9 target mRNA sequence, leading to 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 to allow 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 RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Here, in one aspect, the present invention relates to single-stranded RNA (siRNA) that is produced in cells and promotes the formation of RISC complex that leads to silencing of target gene, i.e., PCSK9 gene. Therefore, the term "siRNA" is also used herein to refer to the above RNAi.

[0083] In another embodiment, the RNAi agent can be a single-stranded siRNA introduced into a cell or organism to inhibit the 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 is described in U.S. Pat. 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 that are 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 a "sense" and an "antisense" orientation to a target RNA, i.e., the PCSK9 gene. In one embodiment of the present invention, the double-stranded RNA (dsRNA) causes the degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0085] Generally, the majority of the nucleotides of each strand of dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides.Furthermore, as used herein, "RNAi agent" may contain ribonucleotides with chemical modifications; RNAi agent may contain substantial modifications in multiple nucleotides.Such modifications may include any type of modification disclosed herein or known in the art.Any such modifications when used in siRNA type molecules are 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 thus linked by a continuous strand of nucleotides between the 3' end of one strand and the 5' end of the other strand that forms the double-stranded structure, the linked RNA strands are called "hairpin loops". When the two strands are covalently linked by means other than a continuous strand of nucleotides between the 3' end of one strand and the 5' end of the other strand that forms the double-stranded structure, the linked structure is called a "linker". The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest strand of the dsRNA minus the overhangs present in the double strand. In addition to the double-stranded structure, the RNAi agent may include 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, e.g., a PCSK9 target mRNA sequence, leading to cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (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 to allow 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 to induce 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 relevant 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 over its entire length, i.e., has no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents that have a nucleotide overhang at one end (i.e., agents that have one overhang and one blunt end) or RNAi agents that have 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 comprises 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, they are generally present in one or more terminal regions, for example, 6, 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end.

[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 skilled in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing. Other conditions may be applied, such as physiologically relevant conditions that may occur inside an organism. For example, complementary sequences are sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi. One skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotides.

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

[0093] "Complementary" sequences as used herein may also include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or 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 from 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 the sequence of the polynucleotide is substantially complementary to a contiguous portion of an mRNA encoding PCSK9.

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

[0097] As used herein, the phrase "inhibiting expression of PCSK9" includes inhibition of expression of any PCSK9 gene (such as, for example, a mouse PCSK9 gene, a rat PCSK9 gene, a monkey PCSK9 gene, or a human PCSK9 gene), 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, e.g., 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 PCSK9 mRNA level, PCSK9 protein level, or 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 (such as a buffer only control or an inactive agent control).

[0100] The phrase "contacting a cell with a double-stranded RNAi agent" as used herein 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. Contact can be performed directly or indirectly. Thus, for example, an RNAi agent can be physically contacted with a cell by performing a method individually, or the RNAi agent can be placed in a situation that allows it to contact a cell later or that allows it to contact a cell later.

[0101] The step of contacting the cells in vitro can be performed, for example, by incubating the cells with the RNAi agent. The step of contacting the cells in vivo can be performed, for example, by injecting the RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another area, bloodstream, or subcutaneous space, so that the RNAi agent will later reach the tissue where the contacted cells are located. For example, the RNAi agent can include and / or be bound to a ligand, such as a GalNAc3 ligand, that directs the RNAi agent to a site of interest, 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, the cells can also be contacted with the 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 non-human animal, preferably a mammal, such as a monkey. Most preferably, the subject or patient is a human.

[0103] "PCSK9-related disease" as used herein is intended to include any disease associated with the PCSK9 gene or protein. Such diseases may be caused, for example, by overproduction of 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 pathologies associated with these disorders, such as heart disease and circulatory system disease.

[0104] A "therapeutically effective amount" as used herein is intended to include an amount of an RNAi agent that, when administered to a patient to treat a disease associated with PCSK9, is sufficient to effect treatment of 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 manner in which the RNAi agent is administered, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by PCSK9 expression, type of previous or concomitant 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 a later-onset disease. A "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 medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the patient being treated.

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

[0107] The term "sample" as used herein includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be from specific organs, parts of organs, or fluids or cells in those organs. In certain embodiments, samples may be from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample from a subject" refers to blood or plasma obtained from a subject. In further embodiments, a "sample 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 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), and uses of such double-stranded RNAi agents.

[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 US 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 the RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of the RNAi agent can be modified completely.The introduction of these motifs interrupts the modification pattern of the sense strand and / or antisense strand, if present.The RNAi agent can be optionally conjugated with GalNAc derivative ligand, for example, on the sense strand.The resulting RNAi agent shows 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 a double-stranded RNAi agent capable of inhibiting 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 range from 12-30 nucleotides in length. For example, each strand can be 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0113] The sense and antisense strands typically form double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The double-stranded region of the RNAi agent can be 12-30 nucleotide pairs long. For example, the double-stranded region can be 14-30 nucleotide pairs long, 17-30 nucleotide pairs long, 27-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-21 nucleotide pairs long, 17-19 nucleotide pairs long, 19-25 nucleotide pairs long, 19-23 nucleotide pairs long, 19-21 nucleotide pairs long, 21-25 nucleotide pairs long, or 21-23 nucleotide pairs long. 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 long.

[0114] In one embodiment, the RNAi agent may 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 may be 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. The overhangs may 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 may form a mismatch with the target mRNA, or the overhangs may be complementary to the targeted gene sequence, or may be another sequence. The first and second strands may also be joined by additional bases, e.g., to form a hairpin, or by other non-basic linkers.

[0115] In one embodiment, each nucleotide in the overhang region of an RNAi agent can be independently a modified or unmodified nucleotide, including, 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 in the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with phosphorothioate between the two nucleotides, where 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 may contain only one overhang, which may enhance the interference activity of RNAi without affecting its overall stability. For example, single-stranded overhang may be located at the 3' end of sense strand or at the 3' end of antisense strand. RNAi may also have a blunt end located at the 5' end of antisense strand (or the 3' end of sense strand) or vice versa. In general, 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 antisense strand and the 3' end overhang of antisense strand favors the introduction of guide strand into RISC process.

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

[0119] In another embodiment, the RNAi agent is a blunt-ended duplex 20 nucleotides in length, where the sense strand contains 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 contains 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, and the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at 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, the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; 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, 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 nucleotides at the end, 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 strand and the antisense strand 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 1-4 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 length of the second strand 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 results in an 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 in the antisense strand.

[0125] In RNAi agents having a double-stranded region 17-23 nucleotides in length, the cleavage sites of the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be 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 counting 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 the RNAi agent may comprise at least one motif of three identical modifications in three consecutive nucleotides at the site of strand break; the antisense strand may have at least one motif of three identical modifications in three consecutive nucleotides at or near the site of strand break.When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand may be aligned such that one motif of three nucleotides in the sense strand and one motif of three nucleotides in the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand base pairs with at least one of the three nucleotides of the motif in the antisense strand.Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0127] In one embodiment, the sense strand of the RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides. The first motif may be 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 that is present in another part of the strand away from a motif at or near the cleavage site of the same strand. The wing modification is 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, and when the motifs are separated by one or more nucleotides, the chemical structures may be the same or different. There may be two or more wing modifications. For example, when there are two wing modifications, each wing modification may be 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 being present at or near the site of strand cleavage. The antisense strand may also contain one or more wing modifications in a sequence similar to 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 in 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 contain 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 aligned such that: two modifications from one strand are each located at one end of the double-stranded region, with 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, with an overlap of one, two, or three nucleotides; two modifications from one strand are located on either side of the lead motif, with 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 the RNAi agent, including nucleotides that are part of a motif, can be modified.Each nucleotide can be modified with the same or different modifications, which can include one or more changes to one or both of the non-linked phosphate oxygen and / or one or more of the linked phosphate oxygen; changes to the components of the ribose sugar, such as the 2' hydroxyl of the ribose sugar; wholesale replacement of the phosphate moiety with a "dephospho" linker; modification or replacement of natural bases; and replacement or modification of the ribose-phosphate backbone.

[0134] Since nucleic acids are polymers of subunits, many of the modifications, such as modifications of bases, or phosphate moieties, or non-linked O of phosphate moieties, are present at repeated positions within the nucleic acid. In some cases, modifications can be present at all of the positions of interest in the nucleic acid, but often this is not the case. For example, modifications can be present only at the 3' or 5' terminal positions, or only at the terminal regions, such as positions on the terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications can be present in double-stranded regions, single-stranded regions, or both. Modifications can be present only in the double-stranded regions of the RNA, or only in the single-stranded regions of the RNA. For example, phosphorothioate modifications at non-linked O positions can be present only at one or both ends, only at the terminal regions, such as positions on the terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of the strand, or in the double-stranded and single-stranded regions, especially at the ends. The 5' end 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 modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribosugar of the nucleobase, with 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 of 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 of 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. 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. The term "alternating motif" as used herein refers to a motif having one or more modifications, each modification occurring at alternating nucleotides of a strand. The alternating nucleotides may 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 may 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, 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 of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0140] In one embodiment, the RNAi agent of the present invention comprises a modification pattern of an alternating motif in the sense strand that is shifted with respect to the modification pattern of the alternating motif 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, whereby there is 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, which pattern first has a shift 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 base pair with the 2'-F modified nucleotides in the antisense strand, and vice versa.The 1st position of the sense strand may start with the 2'-F modification, and the 1st position of the antisense strand may start with the 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 initial 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 the introduction of 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 modifications from the modification of the motif. For example, a portion of a sequence containing a motif may be represented by the phrase "...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 when 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 or antisense strand or both strands at any position of 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 comprise 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 with respect 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 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 bond, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide bond that links 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 are overhang nucleotides, and the third nucleotide is 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 includes mismatches with the target, mismatches within the duplex, or combinations 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 association or dissociation of a particular pairing; the simplest approach is to examine the pair by pair individually, but similar or similar analyses can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; pairings that include universal bases are preferred over canonical pairings.

[0148] In one embodiment, the RNAi agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs in 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 that includes 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 represents, independently, an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide; where 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, YYY are all 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 present at or near the site of cleavage in the sense strand. For example, if the RNAi agent has a double-stranded region 17-23 nucleotides in length, the YYY motif can be present at or near the site of cleavage in the sense strand (e.g., at positions: 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), 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.

[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 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 contains 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 contains 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 represents an oligonucleotide sequence containing independently 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. a can independently represent an oligonucleotide sequence that contains 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 contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0160] In one embodiment, the antisense strand sequence of the RNAi has the 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; Here, 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, when the RNAi agent has a double-stranded region 17-23 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, 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 Each N' 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 N' 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, 1 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 of 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 of 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 5' end and starting from the first paired nucleotide in the double-stranded region; 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 a 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] Each of the sense strands represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an 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 between 14 and 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 ’ represents, independently, 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 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. It is represented 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; 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 an RNAi duplex include the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId)

[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 that independently contains 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 ’ N independently represents an oligonucleotide sequence containing 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 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. Alternatively, at least two of the Z nucleotides are base-paired with a corresponding Z' nucleotide; or all three of the Z nucleotides are 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 to one of the X' nucleotides, alternatively, at least two of the X nucleotides are base-paired to a corresponding X' nucleotide; or all three of the X nucleotides are base-paired to a 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 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 linked to the adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked 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 an adjacent nucleotide via a phosphorothioate bond, and the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[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 an adjacent nucleotide via a phosphorothioate bond, and the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0190] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), the duplexes being linked by a linker. The linker can 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 formula (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 may target the same gene or two different genes; or each of the duplexes may 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 that include one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. In many cases, 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. A ribonucleotide subunit in which the ribose sugar of the subunit is replaced in this manner is referred to herein as a ribose-replacement modified subunit (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, sulfur. The cyclic carrier can be a monocyclic ring system or can include two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring system or can include one or more double bonds.

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

[0199] A. Ligand The double-stranded RNA (dsRNA) agent of the present invention may be optionally conjugated to one or more ligands. The ligand may 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 may 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] where X is O or S. In one embodiment, X is O.

[0201] A variety of entities 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 cell or organ compartment, a tissue, an organ or a region of the body, for example, compared to a species in which such ligand is not present. 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 the lysis of endosomes and / or the transport of the compositions of the 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 the lysis of endosomes and / or the transport of the compositions of the invention, or components thereof, from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptides (Subbarao et al., Biochemistry, 1987, 26:2964-2972), EALA peptides (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586), and derivatives thereof (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 polymeric 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, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or α-helical peptide.

[0207] The ligand may also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody that binds to a specific cell type, such as a renal cell. The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, RGD peptides, RGD peptide mimetics, or aptamers.

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

[0209] A ligand can be a molecule with specific affinity for a protein, e.g., a glycoprotein, or a peptide, e.g., a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, such as a cancer cell, an endothelial cell, or a bone cell. 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, lipopolysaccharide, an activator of MAP kinase 38, or an activator 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 cytoskeleton, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0211] Ligands may increase oligonucleotide uptake into cells, for example, by activating an inflammatory response. Exemplary ligands that may 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 is preferably bound to serum protein, for example, human serum albumin (HSA). HSA-binding ligand allows the distribution of conjugate to target tissues in the body, for example, non-renal target tissues. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligand can be used to (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport to target cell or cell membrane, and / or (c) adjust binding to serum protein, for example, HSA.

[0213] Lipid-based ligand can be used to regulate, for example, control, the binding of conjugate to target tissue.For example, lipid or lipid-based ligand that binds stronger to HSA is less likely to be targeted to kidney, and therefore less likely to be removed from the body.Lipid or lipid-based ligand that binds weaker to HSA can be used to target conjugate to kidney.

[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 is preferably distributed to the kidney. Other moieties that target renal cells can be used in place of or in addition to the lipid-based ligand.

[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 treating disorders characterized by, e.g., malignant or non-malignant unwanted cell proliferation, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low density lipoprotein (LDL), and high density lipoprotein (HDL).

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

[0218] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting 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., 2003). al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic attached to the iRNA agent via an incorporated monomer unit is a peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or an RGD mimetic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids.The peptide moiety may have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below may be used. The RGD peptide moiety may 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). The RGD peptide may facilitate targeting of the iRNA agent to tumors in a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide facilitates targeting of the iRNA agent to the kidney. The RGD peptide may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a particular tissue. For example, a glycosylated RGD peptide may target the iRNA agent to a. 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, as well as synthetic RGD mimetics can be used. In addition to RGD, other moieties that target integrin ligands can be used. In general, 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 a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A peptide that penetrates a microbial cell can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). A cell-penetrating peptide can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[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 may be used to provide additional H-bonds to stabilize the helix. Stability may 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 may 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 replaced by one or more urea, thiourea, carbamate, or sulfonylurea bonds, i.e., peptide bonds; or cyclic peptides.

[0222] The targeting ligand can be any ligand capable of targeting a specific receptor. Examples are folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. Clusters are combinations 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. Ligands can also be based on nucleic acids, for example aptamers. Aptamers can be unmodified or 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 charges, dendrimers with masked or unmasked cationic or anionic charges.

[0224] PK regulators refer to pharmacokinetic modulators. PK regulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK regulators 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 bonds are also known to bind to serum proteins, and therefore short oligonucleotides, such as oligonucleotides of about 5, 10, 15, or 20 bases, containing multiple phosphorothioate bonds in the backbone are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention.

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

[0226] Other ligand conjugates suitable for the present invention are described in U.S. patent application Ser. No. 10 / 916,185, filed Aug. 10, 2004; U.S. patent application Ser. No. 10 / 946,873, filed Sep. 21, 2004; U.S. patent application Ser. No. 10 / 833,934, filed Aug. 3, 2007; U.S. patent application Ser. No. 11 / 115,989, filed Apr. 27, 2005, and U.S. patent application Ser. No. 11 / 944,227, filed Nov. 21, 2007, which are incorporated by reference in their entireties for all purposes.

[0227] When there are two or more ligands, 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, may have endosomolytic activity, or may have PK modulating properties.In a preferred embodiment, all ligands have different properties.

[0228] The ligand may be attached to the oligonucleotide at various positions, e.g., the 3' end, the 5' end, and / or at an internal position. In a preferred embodiment, the ligand is attached to the oligonucleotide via an intervening tether, e.g., a carrier as described herein. The ligand or linking ligand may be present on the monomer as the monomer is incorporated into the growing chain. In some embodiments, the ligand may be incorporated by attachment to a "precursor" monomer after the "precursor" monomer has been incorporated into the growing chain. For example, a monomer having an amino-terminated tether (i.e., no ligand attached), e.g., TAP-(CH2) may be attached to the oligonucleotide. nNH2 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 coupling the electrophilic group of the ligand with the terminal electrophilic group of the tether of the precursor monomer.

[0229] In another example, monomers bearing a chemical group suitable for participating in a click chemistry reaction can be incorporated into the 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, for example, an alkyne or azide, can be attached to the precursor monomer by linking the alkyne and azide together.

[0230] In the case of double-stranded oligonucleotides, 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 bond of the nucleic acid molecule. Conjugation to the purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is bound to the conjugate moiety. Conjugation to the pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-position of the pyrimidine nucleobase can be replaced with a conjugate moiety. Conjugation to the sugar moiety of the nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be bound to the conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be bound to the conjugate moiety, such as in an abasic residue. The internucleoside bond can also have a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiesters, phosphorothioates, phosphorodithioates, phosphoramidates, 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, eg, a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide.

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

[0234] In one embodiment, the dsRNA of the present invention is conjugated to bivalent and trivalent branched linkers comprising a structure as 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), CH2, CH2NH, or CH2O; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently for each occurrence absent, alkylene, or substituted alkylene, where one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5Care each independently absent for each occurrence, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each, independently for each occurrence, is a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; R a is H or an amino acid side chain.

[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, such as a subject suffering from a lipid disorder such as dyslipidemia), can be accomplished in a number of 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 comprising an iRNA, e.g., a dsRNA, to the subject. Alternatively, in vivo delivery can be accomplished indirectly, by administering one or more vectors that encode and direct the expression of the iRNA. Examples of these alternatives are described further below.

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

[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, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A. et al., International PCT Publication No. WO 00 / 22113, Conrad, International PCT Publication No. WO 00 / 22114, and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a matter of hours to weeks) or sustained (weeks to months or longer), depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. 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 strand or strands of the iRNA can be transcribed from a promoter in an expression vector. Two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell, where two separate strands are expressed to produce, for example, dsRNA. Alternatively, each individual strand of the dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence to have a stem-loop structure.

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

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

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

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

[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 the host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to the patient. Further details on retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references showing the use of retroviral vectors in gene therapy are Clowes et al., 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, 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 the iRNA of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes, for example, to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia and cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 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 iRNA 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 iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

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

[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 made to target different cells by engineering the vector to express different capsid protein serotypes. See, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[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 pharma- ceutically acceptable carrier is also provided herein. The pharmaceutical composition containing the iRNA is useful for treating diseases or disorders associated with the expression or activity of the PCSK9 gene, 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., by 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] The pharmaceutical composition comprising the RNAi agent of the present invention may be, for example, a solution with or without a buffer, or a composition containing a pharma- ceutically acceptable carrier.Such compositions include, 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, for example, saline or water. Alternatively, the free siRNA can also be administered in a suitable buffer. The buffer can include 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 further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, for example, the physiologic value of human plasma. Solutes that can be added to the buffer 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 composition of the present invention can be administered at a dosage sufficient to inhibit the expression of the PCSK9 gene. In general, the suitable dosage of the iRNA of the present invention is in the range of about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient 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 concentration 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 25 to about 50 mg / kg, about 30 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 50 to about 50 mg / kg, about 50 to about 50 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 / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present invention.

[0260] For example, the RNAi agent, e.g., dsRNA, may have a concentration of about 0..01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.8, 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, 10, 10, 10, 10, 10, 10, 10, 10, 10 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, for example, 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 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 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 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 50 to about 50 mg / kg, about 50 to about 50 mg / kg, about 50 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 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 intended to be part of the present invention.

[0262] For example, about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3 ,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4 ,8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 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 a day, or the iRNA can be administered as two, three or more subdoses at appropriate intervals throughout the day, or even using delivery via continuous infusion or sustained release formulations. In that case, each subdose should contain a correspondingly smaller amount of iRNA to achieve the total daily dosage. The dosage unit can also be formulated for delivery over several days, for example using a conventional sustained release formulation that provides sustained release of the iRNA over a period of several days. Sustained release formulations are well known in the art and can be used with the agents of the invention, as they are particularly useful for delivering agents to specific sites. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0264] In other embodiments, a single dose of the pharmaceutical composition can last for a long time, with subsequent doses being administered at intervals of 3, 4, or 5 days or less, or at intervals of 1, 2, 3, or 4 weeks or less. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered bi-monthly.

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

[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 to determine the effective dose for treatment.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 site to be treated. Administration can be topical (e.g., by transdermal patch), pulmonary administration, for example, by inhalation or insufflation of powders or aerosols, such as by nebulizers; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous administration, for example, by implantation device; or intracranial administration, for example, by intraparenchymal, intrathecal or intraventricular administration.

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

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

[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. The RNAi agent preparation is then added to the micelles containing the lipid components. The cationic groups in the lipids interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposomal formulation of the RNAi agent.

[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 generating stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as a component of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described 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 (e.g., Mayer et al., Endocrinol. 115:757, 1984). Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw and extrusion (see, for example, 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 package 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 transferred 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] Liposomes that are pH-sensitive and negatively charged entrap nucleic acids rather than complexing with them. Because both the nucleic acid and the lipid are similarly charged, repulsion occurs rather than complexation. Nevertheless, some nucleic acids are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., 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 dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleyl phosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

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

[0279] Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, provide enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) that are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes results from reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., 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) report the use of monosialoganglioside G to improve the blood half-life of liposomes. M1 (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G to inhibit the cellular cellular endothelial function of sphingomyelin. M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

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

[0283] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of tissue culture cell membranes, resulting in delivery of RNAi agents (see, e.g., Felgner, PL et al., 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] A DOTMA analog, 1,2-bis(oleyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously attach to negatively charged cell surfaces, fuse with cell membranes, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are attached by ester rather than ether bonds.

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

[0286] Another cationic lipid conjugate involves derivatization of lipids with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made 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 formulations are particularly suitable for topical administration, and liposomes exhibit several advantages over other formulations.These advantages include the reduced side effects associated with the high systemic absorption rate of the administered drug, the increased accumulation of the administered drug in the desired target, and the ability to administer RNAi agents to the skin.In some implementations, liposomes are used to deliver RNAi agents to epidermal cells and to promote the penetration of RNAi agents into dermal tissues, such as skin.For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been reported (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, RM and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

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

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

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

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

[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. In general, their HLB values ​​range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

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

[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 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.A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure, so that all the hydrophobic parts of the molecule face inward, and the hydrophilic parts remain in contact with the surrounding aqueous phase.When the environment is hydrophobic, the reverse arrangement exists.

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

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

[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 formation of the mixed micelle composition.

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

[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 at least by a factor of two or three, over 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] The term "LNP" as used herein refers to a stable nucleic acid-lipid particle. LNPs typically include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs have an extended circulation life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the site of administration), making them extremely useful for systemic applications. LNPs include "pSPLPs," which include encapsulated condensing agent-nucleic acid complexes, as shown in PCT Publication No. WO 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, the nucleic acid, when present in the nucleic acid-lipid particles of the present invention, is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. 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 to be part of the invention.

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

[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) with 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), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleyl-phosphatidylethanolamine 4-(N The lipid may be an anionic or neutral lipid, including 1-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid, when cholesterol is included, may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[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 mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents particle aggregation can be 0 mol% to about 20 mol%, or 2 mol% of the total lipid present in the particle.

[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, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Respective stock solutions in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml, PEG-Ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-Ceramide C16 stock solutions can then be combined, for example, in a molar ratio of 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., in sodium acetate (pH 5)) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles usually form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using, for example, a thermobarrel extruder such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged, for example, with phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. [ka]

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

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

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

[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 the methods described in more detail in the Examples. All substituents are as defined below unless otherwise indicated.

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

[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 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 may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be bonded via any heteroatom or carbon atom. Heterocycle includes heteroaryl as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[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, -NRxSORy, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy, where n is 0, 1 or 2, and Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORx, heterocycle, -NRxRy, -NRxC(=O)Ry, -NRxSORy, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SONRx and -SONNRxRy.

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

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

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

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

[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 The preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid, followed by dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent was removed on a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. The 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), a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF was slowly added at 00 C under nitrogen atmosphere. After complete addition, the reaction mixture was allowed to warm to room temperature and then heated to reflux for 4 h. The progress of the reaction was monitored by TLC. After completion of the reaction (by TLC), the mixture was cooled to 00 C and quenched by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 h and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. The volatiles were removed under reduced pressure to give the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1 H-NMR (DMSO, 400MHz): δ = 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66~2.60 (m, 2H), 2.50~2.45 (m, 5H).

[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 was added (37.2 mL, 0.2669 mol) and cooled to 0 °C under nitrogen atmosphere. After slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM, the reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC), the mixture was washed successively with 1N HCl solution (1 x 100 mL) and saturated NaHCO3 solution (1 x 50 mL). The organic layer was then dried over anhydrous Na2SO4 and the solvent was evaporated to give the crude material, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11 g (89%). 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 acetone and water (10:1) in a 500 mL one-neck round-bottom flask, to which N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added followed by 4.2 mL of a 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (about 3 h), the mixture was quenched by the addition of solid Na2SO3 and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 x 100 mL), followed by saturated NaHCO3 (1 x 50 mL) solution, water (1 x 30 mL) and finally brine (1 x 50 mL). The organic phase was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. Silica gel column chromatography purification of the crude material gave a mixture of diastereomers, which were separated by preparative HPLC. Yield:-6 g of crude 517A-peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

[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. 1H-NMR (CDCl3, 400 MHz): δ = 7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

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

[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). The particles should be about 20-300 nm in size, for example 40-100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, and the fraction entrapped, are estimated using a dye exclusion assay. Samples of the formulated dsRNA may be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disintegrating surfactant, for example 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant against a standard curve. The 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, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. 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] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or mini-tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desired. In some embodiments, oral formulations are those in which the dsRNA characterizing the invention is administered with one or more permeation enhancer surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride, or pharma- ceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as fatty acid / salts in combination with bile acids / salts. One exemplary combination is the sodium salts of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The DsRNA characterizing the present invention can be delivered orally in granular form, including spray-dried particles, or complexed to form micro- or nanoparticles.dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, ... Oral formulations for dsRNA and their formulations are described in U.S. Pat. No. 6,887,906, U.S. Patent Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[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 pharma- ceutically acceptable carriers or excipients.

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

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

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

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

[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 means of emulsifiers or through the viscosity of the formulation.Either of the phases of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams.Other means of stabilizing emulsions include the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

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

[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 incorporate water to form w / o emulsions, yet maintain their semi-solid consistency. Finely divided solids are used as good emulsifiers, especially in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

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

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

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

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

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

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

[0359] Surfactants used in the preparation of microemulsions, alone or in combination with cosurfactants, include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750). The cosurfactants, which are usually short chain alcohols such as ethanol, 1-propanol, and 1-butanol, serve to increase the interfacial fluidity by penetrating the surfactant film and thus forming an irregular film due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can include materials such as, but not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

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

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

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

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

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

[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 the iRNA through mucosa. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether) (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorinated emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[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), as well as their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0367] The physiological roles of bile include facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). A variety of natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharma- ceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee, J. Am. Chem. Soc. 1999, 144:1311-1323, 2002; Lee, J. Am. Chem. Soc. 1999, 144:1311-1323, 2002). et al.,Critical Reviews in Therapeutic Drug Carrier Systems,1991,page 92;Swinyard,Chapter 39 In:Remington's Pharmaceutical Sciences,18th Ed.,Gennaro,ed.,Mack Publishing Co.,Easton,Pa.,1990,pages 782-783;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

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

[0369] As used herein, non-chelating, non-surfactant penetration enhancers may be defined as compounds that exhibit little activity as chelators or surfactants, but nevertheless enhance the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0370] Agents that enhance the uptake of iRNA at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids such as lipofectin (Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., PCT Application WO 97 / 30731) are also known to enhance the cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), RNAiMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;(Madison, WI), TransFast (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 FECTOR vectors include RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA).

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

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

[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 predetermined components of the pharmaceutical composition, taking into account the planned method of administration. Typical pharmaceutical carriers include, but are not limited to, binders (such as, for example, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as, for example, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate); lubricants (such as, for example, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); tablet disintegrants (such as, for example, starch, sodium starch glycolate, and the like); and wetting agents (such as, for example, sodium lauryl sulfate, and the like).

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

[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. The solutions can also contain buffers, diluents, and other suitable additives. Suitable organic or inorganic excipients that are pharma- ceutically acceptable for parenteral administration and do not adversely react with nucleic acids can be used.

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

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

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

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

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

[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 results observed, using standard measures of efficacy known in the art or described herein.

[0383] IV. METHODS OF INHIBITORY PCSK9 EXPRESSION The present invention provides a method for inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell. The method includes contacting a 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 the cell with the double-stranded RNAi agent can be performed in vitro or in vivo. The step of contacting the cell with the 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 contact can be performed directly or indirectly, as described above. Furthermore, the step of contacting the cell can be performed 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 site of interest, for example, the liver of a subject.

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

[0386] The phrase "inhibiting expression of PCSK9" as used herein is intended to refer to the inhibition of expression of any PCSK9 gene (such as, for example, mouse PCSK9 gene, rat PCSK9 gene, monkey PCSK9 gene, or human PCSK9 gene), as well as 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.

[0387] "Inhibiting expression of the PCSK9 gene" includes any level of inhibition of the PCSK9 gene, e.g., 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, e.g., PCSK9 mRNA level, PCSK9 protein level, or lipid level. The level can be assessed in an individual cell or a group of cells, including, for example, 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 associated with 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, such as a buffer only control or an inactive agent control.

[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 have been treated such 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 are substantially identical to the first cell or group of cells but have 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 reducing 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, either 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 expression of PCSK9 protein is manifested in a reduction in the level of PCSK9 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived from a subject). As described above for assessing mRNA suppression, inhibition of expression levels of protein in treated cells or cell groups can similarly be expressed as a percentage of the level of protein in a control cell or cell group.

[0393] A control cell or group of cells that can be used to evaluate inhibition of expression of the PCSK9 gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the present invention. For example, a control cell or group of cells is 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 to assess mRNA expression. In one embodiment, the level of expression of PCSK9 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 the use of PAX gene (PreAnalytix, Switzerland). Exemplary 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. The term "probe" as used herein 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 suitable 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 includes 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 (experimental embodiment described in Mullis, 1987, 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, nucleic acid amplification and / or reverse transcriptase processes of mRNA in a sample (to prepare cDNA), followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for detection of nucleic acid molecules when they are present in very low numbers. In certain embodiments of the present invention, the level of expression of PCSK9 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 that contains bound nucleic acid).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.Measurement of PCSK9 expression level can also include the use of nucleic acid probes in solution.

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

[0400] The expression level of PCSK9 protein can be measured by any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reaction, spectrophotometric analysis, 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] The term "sample" as used herein includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, lymphatic fluid, urine, cerebrospinal fluid, saliva, ocular fluid, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be from specific organs, parts of organs, or fluids or cells in those organs. In certain embodiments, samples may be from the liver (e.g., the whole liver or specific parts of the liver, or specific types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample from a subject" refers to blood or plasma obtained from a subject. In further embodiments, a "sample 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 the subject so that the RNAi agent is delivered to a specific site in the subject. The inhibition of PCSK9 expression can be evaluated using the measurement of the level or change in the level of PCSK9 mRNA or PCSK9 protein in a sample derived from a 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 a method for treating or preventing diseases and conditions that can be regulated by downregulating PCSK9 gene expression. For example, the compositions described herein can be used to treat other forms of lipid imbalance, such as hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, and conditions associated with these disorders, such as heart disease and circulatory system disease. Other diseases and conditions that can be regulated by downregulating PCSK9 gene expression include lysosomal storage diseases, including, but not limited to, Niemann-Pick disease, Tay-Sachs disease, lysosomal acid lipase deficiency, and Gaucher disease. The method includes administering a therapeutically or prophylactically effective amount of the RNAi agent of the present invention to a subject. In an embodiment, the method includes administering an effective amount of PCSK9 siRNA to a patient with a heterozygous LDLR genotype.

[0404] The effect of the decreased PCSK9 gene preferably results in a reduction in LDLc (low density lipoprotein cholesterol) levels in the mammal's blood, and more particularly in serum, hi certain 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, a 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 release RNAi agent continuously 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, the administration is by pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, intraarterial, 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 methods of administration include epidural, intracerebral, intraventricular, intranasal, intraarterial, intracardiac, intraosseous, intrathecal, and intravitreal, and pulmonary. The method of administration can be selected based on whether local or systemic treatment is desired and based on the site to be treated. The route and site of administration can be selected to facilitate targeting.

[0411] The methods include, for example, administering an iRNA agent at a dose sufficient to reduce the level of PCSK9 mRNA for at least 5, and 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, and more preferably 7, 10, 14, 21, 25, 30, or 40 days after the first, single dose is administered, 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 4 weeks, no more than once every 3 weeks, no more than once every 2 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] In general, 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% of 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 amount 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 for a period of time, such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. Administration may be repeated periodically, such as, for example, every other week (i.e., every two weeks) for one month, two months, three months, four months, or more. After an initial treatment regimen, the treatment agent may be administered less frequently. For example, after three months of administration every other week, administration may be repeated once a month for six months or one year, or 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] Prior to administering a full dose of the iRNA agent, the patient may be administered a smaller dose (such as less than 5% infusion reactions) 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 increased cytokine (e.g., TNF-α or INF-α) levels.

[0417] The efficacy of treatment or prevention is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when a worsening or otherwise expected symptom does not occur. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more, in a measurable parameter of the disease may indicate an effective treatment. The efficacy of a particular iRNA agent of the invention, or a given formulation of the iRNA agent, may also be determined using an experimental animal model for a given disease, as known in the art. When using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant decrease in a marker or symptom is observed.

[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 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, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 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, about 50 mg / kg, about 51 mg / kg, about 52 mg / kg, about 53 mg / kg, about 54 mg / kg, about 55 mg / kg, about 56 mg / kg, about 57 mg / kg, about 58 mg / kg, about 59 mg / kg, about 60 mg / kg, about 61 mg / kg, about 62 g, 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. In one embodiment, the iRNA agent is administered at a dose of about 25 mg / kg.

[0420] The dose of an RNAi agent that may be administered to a subject can be adjusted to balance the risks and benefits of a particular dose, for example, to obtain 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. When it is necessary to facilitate repeated or frequent infusion, implantation of a delivery device, such as a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracapsular or intracapsular), or a reservoir may be desirable. In some embodiments, the number or amount of subsequent administrations depends on the achievement of a desired effect, such as suppression of the PCSK9 gene, or the achievement of a therapeutic or preventive effect, such as a reduction in 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 twelve 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 longer periods during which the RNAi agent is not administered. For example, the regimen includes an initial set of doses administered at a relatively short time period (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 time 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 the achievement of a 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 once a week at a dose of 0.5 mg / kg. In one embodiment, the iRNA agent is administered once a week at a dose of 1 mg / kg. In another embodiment, the iRNA agent is administered once a week at a dose of 2 mg / kg.

[0425] In certain embodiments, the RNAi agent is administered in a dosing regimen that includes an "initial administration phase" of closely spaced administrations, followed possibly by a "maintenance phase" in which the RNAi agent is administered at longer intervals. In one embodiment, the initial administration phase includes five daily doses of the RNAi agent for the first week. In another embodiment, the maintenance phase includes one or two weekly doses of the RNAi agent. In a further embodiment, the maintenance phase lasts for five weeks. In one embodiment, the initial administration phase includes five weekly doses of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg. In another embodiment, the maintenance phase includes one, two, or three weekly doses of 2 mg / kg, 1 mg / kg, or 0.5 mg / kg, once per week, once per two months, once per three months, once per four months, once per five months, or once per 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 the 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 combination vasoprotectant (e.g., AGI-1067 from Atherogenics), a glycoprotein Ilb / IIIa inhibitor, an aspirin or 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., Befizal® / Cedur® / Bezalip® from Roche, Bezatol from Kissei), clofibrate (e.g., Atromid-S® from Wyeth), fenofibrate (e.g., Lipidil / Lipantil from Fournier, Tricor® from Abbott, Lipantil from Takeda, generics), gemfibrozil (e.g., Lopid / Lipur from Pfizer), and ciprofibrate (Modalim® from Sanofi-Synthelabo).Exemplary bile acid sequestrants include, for example, cholestyramine (Questran® and Questran Light™ from Bristol-Myers Squibb), colestipol (e.g., Colestid from Pharmacia), and colesevelam (WelChol™ from Genzyme / Sankyo). Exemplary niacin treatments include, for example, immediate release formulations such as Nicobid from Aventis, Niacor from Upsher-Smith, Nicolar from Aventis, and Perycit from Sanwakagaku. Sustained release formulations of niacin include, for example, Niaspan from Kos Pharmaceuticals and SIo-Niacin from Upsher-Smith. Exemplary antiplatelet agents include, for example, aspirin (e.g., Aspirin from Bayer), clopidogrel (Plavix from Sanofi-Synthelabo / Bristol-Myers Squibb), and ticlopidine (e.g., Ticlid from Sanofi-Synthelabo and Panaldine from Daiichi). Other aspirin-like compounds useful in combination with dsRNA targeting PCSK9 include, for example, Asacard (extended-release aspirin from Pharmacia) and pamicogrel (Kanebo / Angelini Ricerche / CEPA). Exemplary angiotensin-converting enzyme inhibitors include, for example, ramipril (e.g., Altace from Aventis) and enalapril (e.g., Vasotec from Merck & Co.). 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 called 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 modulating 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), ApoAl (UCB Pharma / Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-Al (ABCA1) (CV Therapeutics / Incyte, Aventis, Xenon). Exemplary glycoprotein Ilb / IIIa inhibitors include, for example, roxifiban (also called 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 Nycline (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 Sci. 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 an 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 being in need of LDL reduction, LDL reduction without HDL reduction, ApoB reduction, or total cholesterol reduction. The method includes, for example, administering to the patient an siRNA in an amount sufficient to reduce the patient's LDL or ApoB levels without substantially reducing HDL levels.

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

[0434] Before prescribing or administering the iRNA agent of the present invention, a medical care provider such as a doctor, a nurse, or a family member can take a family history.Furthermore, a test can be performed to determine genotype or phenotype.For example, before PCSK9 dsRNA is administered to a patient, a DNA test can be performed on a sample, such as a blood sample, from the patient to identify PCSK9 genotype and / or phenotype.In another embodiment, a test is performed to identify related genotype and / or phenotype, such as LDLR genotype. 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(l): 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., instructions 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 effective amount or a prophylactically effective amount.

[0436] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Similar or equivalent methods and materials as described herein can be used to practice or test the iRNA and methods that characterize the present invention, and suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting. The present disclosure relates, for example, to the following: [Section 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 Nb 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 said 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: [Section 2] The double-stranded RNAi agent according to item 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. [Section 3] Item 2. The double-stranded RNAi agent according to item 1, wherein k is 0; l is 0; k is 1; l is 1; k and l are both 0; or k and l are both 1. [Section 4] The double-stranded RNAi agent of paragraph 1, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'. [Section 5] Item 2. The double-stranded RNAi agent according to Item 1, wherein the YYY motif is present at or near the cleavage site of the sense strand. [Section 6] Item 2. The double-stranded RNAi agent according to Item 1, wherein the Y'Y'Y' motif is present at positions 11, 12, and 13 of the antisense strand at the 5' end. [Section 7] 7. The double-stranded RNAi agent according to claim 6, wherein Y' is 2'-O-methyl. [Section 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 according to item 1, wherein the double-stranded RNAi agent is represented by: [Section 9] Formula (III) may be replaced by 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) The double-stranded RNAi agent according to item 1, wherein the double-stranded RNAi agent is represented by: [Section 10] Formula (III) may be replaced by 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) The double-stranded RNAi agent according to item 1, wherein the double-stranded RNAi agent is represented by: [Section 11] Formula (III) may be represented by formula (IIId): Sense:5'np -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, a and N a ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides. The double-stranded RNAi agent according to item 1, wherein the double-stranded RNAi agent is represented by: [Section 12] Item 2. The double-stranded RNAi agent according to Item 1, wherein the double-stranded region is 15 to 30 nucleotide pairs in length. [Section 13] Item 13. The double-stranded RNAi agent according to Item 12, wherein the double-stranded region is 17 to 23 nucleotide pairs in length. [Section 14] Item 13. The double-stranded RNAi agent according to Item 12, wherein the double-stranded region is 17 to 25 nucleotide pairs in length. [Section 15] Item 13. The double-stranded RNAi agent according to Item 12, wherein the double-stranded region is 23 to 27 nucleotide pairs in length. [Section 16] Item 13. The double-stranded RNAi agent according to Item 12, wherein the double-stranded region is 19 to 21 nucleotide pairs in length. [Section 17] Item 13. The double-stranded RNAi agent according to Item 12, wherein the double-stranded region is 21 to 23 nucleotide pairs in length. [Section 18] Item 2. The double-stranded RNAi agent according to Item 1, wherein each strand has 15 to 30 nucleotides. [Section 19] The double-stranded RNAi agent according to item 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. [Section 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. [Section 21] 2. The double-stranded RNAi agent according to claim 1, wherein the ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker. [Section 22] The ligand is [C1] TIFF0007670747000019.tif63161 Item 2. The double-stranded RNAi agent according to Item 1, [Section 23] Item 2. The double-stranded RNAi agent according to Item 1, wherein the ligand is attached to the 3' end of the sense strand. [Section 24] Schematic diagram below [C2] TIFF0007670747000020.tif71161 (wherein X is O or S). [Section 25] The double-stranded RNAi agent according to paragraph 1, further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage. [Section 26] 26. The double stranded RNAi agent of paragraph 25, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. [Section 27] 27. The double-stranded RNAi agent of claim 26, wherein the strand is the antisense strand. [Section 28] 27. The double-stranded RNAi agent of claim 26, wherein the strand is the sense strand. [Section 29] 26. The double stranded RNAi agent of paragraph 25, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. [Section 30] 30. The double stranded RNAi agent of claim 29, wherein the strand is the antisense strand. [Section 31] 30. The double stranded RNAi agent of claim 29, wherein the strand is the sense strand. [Section 32] 26. The double stranded RNAi agent of paragraph 25, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand. [Section 33] 33. The double-stranded RNAi agent of claim 32, wherein the strand is the antisense strand. [Section 34] Item 2. The double-stranded RNAi agent according to Item 1, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair. [Section 35] The double-stranded RNAi agent of paragraph 1, wherein the Y nucleotide comprises a 2'-fluoro modification. [Section 36] The double-stranded RNAi agent of paragraph 1, wherein the Y' nucleotide comprises a 2'-O-methyl modification. [Section 37] The double-stranded RNAi agent according to item 1, wherein p'>0. [Section 38] Item 2. The double-stranded RNAi agent according to item 1, wherein p'=2. [Section 39] 39. The double-stranded RNAi agent of claim 38, wherein q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA. [Section 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. [Section 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. [Section 42] At least one n p 42. The double-stranded RNAi agent according to any one of claims 37 to 41, wherein the ' is linked to an adjacent nucleotide via a phosphorothioate bond. [Section 43] All n p 43. The double-stranded RNAi agent of claim 42, wherein the nucleotides A, B, C, and D are linked to adjacent nucleotides via phosphorothioate bonds. [Section 44] The double-stranded RNAi agent according to paragraph 1, selected from the group of RNAi agents listed in Table 1, Table 2, Table 9, Table 10, Table 12, and FIG. 12A. [Section 45] The double-stranded RNAi agent according to item 1, selected from the group consisting of AD-53815, AD-56663, AD-56658, AD-56676, AD-56666, AD-57928, and AD-60212. [Section 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 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, said modifications being 2'-O-methyl or 2'-fluoro modifications; N b the modification on Y is different from said 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: [Section 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 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 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 said 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: [Section 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 -Nb '-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 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 said 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: [Section 49] 1. A double-stranded RNAi agent capable of inhibiting expression of proprotein convertase subtilisin kexin 9 (PCSK9) in a cell, the double-stranded R...

Claims

1. 1. A double-stranded RNAi agent comprising a sense strand complementary to an antisense strand forming a double-stranded region, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length; the antisense strand consists of the nucleotide sequence of SEQ ID NO: 1666: 5'-ACAAAAGCAAAAACAGGUCUAGAA-3'; the sense strand consists of the nucleotide sequence of SEQ ID NO: 197 5'-CUAGACCUGUUUUGCUUUGU-3' or the nucleotide sequence of SEQ ID NO: 1665 5'-CUAGACCUGUTUUGCUUUUGU-3'; the antisense strand comprises a two nucleotide overhang at the 3' end; each of the nucleotides of the sense strand and each of the nucleotides of the antisense strand independently comprises a nucleotide modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 2'-deoxythymidine (dT) modification; the sense strand and the antisense strand each independently comprise at least one motif of three identical modifications on three consecutive nucleotides, the modifications comprising a 2'-O-methyl modification or a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at the 3'-end and two phosphorothioate internucleotide linkages between the three terminal nucleotides at the 5'-end; the sense strand comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at the 5'-end, and the sense strand is conjugated to a ligand that is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; Double-stranded RNAi agents.

2. 2. The double-stranded RNAi agent of claim 1, wherein the sense strand consists of the nucleotide sequence 5'-CUAGACCUGUTUUGCUUUUGU-3' of SEQ ID NO:1665.

3. 2. The double-stranded RNAi agent of claim 1, wherein the antisense strand consists of the nucleotide sequence of SEQ ID NO:1666 5'-ACAAAGCAAAACAGGUCUAGAA-3' and the sense strand consists of the nucleotide sequence of SEQ ID NO:1665 5'-CUAGACCUGUTUUGCUUUUGU-3'.

4. The double-stranded RNAi agent of claim 1, wherein the antisense strand consists of the nucleotide sequence of SEQ ID NO: 1666, 5'-ACAAAGCAAAACAGGUCUAGAA-3', and the sense strand consists of the nucleotide sequence of SEQ ID NO: 197, 5'-CUAGACCUGUUUUGCUUUUGU-3'.

5. The ligand is 【Chemistry 1】 The double-stranded RNAi agent according to any one of claims 1 to 4,

6. The double-stranded RNAi agent of any one of claims 1 to 5, wherein the ligand is attached to the 3' end of the sense strand.

7. Schematic diagram below 【Chemistry 2】 7. The double-stranded RNAi agent of claim 6, conjugated to the ligand of the formula:

8. A pharmaceutical composition comprising the double-stranded RNAi agent according to any one of claims 1 to 7.

9. The pharmaceutical composition of claim 8 further comprising a buffer.

10. 10. The pharmaceutical composition of claim 9, wherein the buffer comprises an acetate buffer, a citrate buffer, a prolamine buffer, a carbonate buffer, or a phosphate buffer, or any combination thereof.

11. The pharmaceutical composition of claim 8 further comprising a non-buffered solution.

12. The pharmaceutical composition of claim 11 , wherein the non-buffered solution is saline or water.

13. An isolated cell containing the double-stranded RNAi agent of any one of claims 1 to 7.

14. 1. A method for inhibiting PCSK9 expression in a cell in vitro, comprising: (a) contacting said cell with the double-stranded RNAi agent according to any one of claims 1 to 7 or the pharmaceutical composition according to any one of claims 8 to 12; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the PCSK9 gene, thereby inhibiting expression of the PCSK9 gene in the cells; The method includes:

15. 15. The method of claim 14, wherein the PCSK9 expression is inhibited by at least 30%.

16. The double-stranded RNAi agent according to any one of claims 1 to 7 or the pharmaceutical composition according to any one of claims 8 to 12 for use in a method for treating a subject suffering from a disorder mediated by PCSK9 expression.

17. 17. The double-stranded RNAi agent or pharmaceutical composition of claim 16, wherein the subject is a human.

18. 20. The double-stranded RNAi agent or pharmaceutical composition of claim 17, wherein the human suffers from hypercholesterolemia.

19. 19. The double-stranded RNAi agent or pharmaceutical composition of any one of claims 16 to 18, wherein the double-stranded RNAi agent is suitable for administration at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg.

20. 17. The double-stranded RNAi agent or pharmaceutical composition of claim 16, wherein the double-stranded RNAi agent is suitable for administration in two or more doses.

21. 21. The double-stranded RNAi agent or pharmaceutical composition of claim 20, wherein the double-stranded RNAi agent is suitable for administration in a dosing regimen comprising an initial administration phase followed by a maintenance phase.

22. The double-stranded RNAi agent or pharmaceutical composition of claim 21, wherein the double-stranded RNAi agent is suitable for administration once every three months during a maintenance phase.

23. The double-stranded RNAi agent or pharmaceutical composition of claim 21, wherein the double-stranded RNAi agent is suitable for administration once every six months during a maintenance phase.

24. The double-stranded RNAi agent or pharmaceutical composition according to any one of claims 16 to 23, wherein the composition is suitable for subcutaneous administration.

25. The double-stranded RNAi agent or pharmaceutical composition of any one of claims 16 to 23, wherein the composition is suitable for intravenous administration.

26. The double-stranded RNAi agent according to any one of claims 1 to 7 or the pharmaceutical composition according to any one of claims 8 to 12 for use in a method for treating hypercholesterolemia in a subject.

27. 27. The double-stranded RNAi agent or pharmaceutical composition of claim 26, wherein the subject is a human.

28. 28. The double-stranded RNAi agent or pharmaceutical composition of claim 26 or 27, wherein the double-stranded RNAi agent is suitable for administration at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg.

29. 27. The double-stranded RNAi agent or pharmaceutical composition of claim 26, wherein the double-stranded RNAi agent is suitable for administration in two or more doses.

30. 30. The double-stranded RNAi agent or pharmaceutical composition of Claim 29, wherein the double-stranded RNAi agent is suitable for administration in a dosing regimen comprising an initial administration phase followed by a maintenance phase.

31. The double-stranded RNAi agent or pharmaceutical composition of claim 30, wherein the double-stranded RNAi agent is suitable for administration once every three months during a maintenance phase.

32. The double-stranded RNAi agent or pharmaceutical composition of claim 30, wherein the double-stranded RNAi agent is suitable for administration once every six months during a maintenance phase.

33. The double-stranded RNAi agent or pharmaceutical composition of any one of claims 26 to 32, wherein the composition is suitable for subcutaneous administration.

34. The double-stranded RNAi agent or pharmaceutical composition of any one of claims 26 to 32, wherein the composition is suitable for intravenous administration.

35. 27. The double-stranded RNAi agent or pharmaceutical composition of claim 26, which results in a reduction of serum cholesterol in the subject.

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