Apolipoprotein C3 (APOC3) iRNA composition and method of use thereof

Abstract

This provides a regulator of APOC3 expression for treating apolipoprotein C3 (APOC3)-related disorders such as hypertriglyceridemia. [Solution] The present invention provides an RNAi agent that targets the APOC3 gene, such as a double-stranded RNAi agent, a method of using such an RNAi agent to inhibit APOC3 expression, and a method of treating a subject having APOC3-related disorder. In some embodiments, the double-stranded RNAi agent for inhibiting APOC3 expression comprises a sense strand and an antisense strand forming a double-stranded region, each comprising at least 15 consecutive nucleotides that differ from a specific nucleotide sequence by 3 nucleotides or less, substantially all of the nucleotides on at least one of the strands are modified nucleotides, and the sense strand is coupled to a ligand attached to its 3' end.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 080,941, filed on November 17, 2014, and U.S. Provisional Patent Application No. 62 / 136,159, filed on March 20, 2015. The entire contents of each of the foregoing applications are hereby incorporated by reference herein.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety herein. The ASCII copy created on November 17, 2015, has the name 121301 - 02520_SL.txt and a size of 212,0 .

Background Art

[0003] Apolipoprotein C3 (APOC3) is a very - low - density lipoprotein (VLDL) and an important regulator of lipoprotein metabolism. In humans, APOC2 is encoded by the APOC3 gene, which is located as a gene cluster together with the APOA1 and APOA4 genes on the long arm of chromosome 11. APOC3 is expressed as a small 99 - amino - acid protein in the liver and, to a lesser extent, in the intestine. When a 20 - amino - acid signal peptide is removed in the endoplasmic reticulum, a 79 - amino - acid mature ApoC3 protein is formed, which can exist as a non - glycosylated or glycosylated isoform.

[0004] The primary role of APOC3 is as a regulator of lipolysis through non-competitive inhibition of endothelial-bound lipoprotein lipase (LPL). LPL hydrolyzes triacylglycerols in triacylglycerol-rich lipoproteins (TRLs), releasing fatty acids into the plasma and converting large triacylglycerol-rich particles into smaller triacylglycerol-depleted remnant lipoproteins. APOC3-deficient individuals have low TRL levels and exhibit highly efficient lipolysis of triacylglycerols. Furthermore, mice with a genetic deletion of the APOC3 gene have also been shown to have low plasma triacylglycerol levels and efficient TRL catabolism. APOC3 also inhibits hepatic lipase (HL), a lipolytic enzyme with triacylglycerol lipase and phospholipase A1 activity synthesized in the liver. The HL inhibitory effect of APOC3 further reduces hepatic lipolysis and TRL remnant uptake. APOC3 has also been shown to stimulate the synthesis of very low-density lipoprotein (VLDL). The underlying mechanism associated with this APOC3 effect may involve the inhibition of proteasome-mediated APOB degradation, which could lead to increased APOB synthesis and secretion, as well as increased VLDL triacylglycerol synthesis. Therefore, APOC3 may play an important role in regulating VLDL production by the liver.

[0005] Cellular studies have reported that APOC3 may inhibit TRL and remnant binding to lipoprotein receptors in the liver. APOC3 can eliminate APOB-mediated and ApoE-mediated lipoprotein binding to low-density lipoprotein receptors (LDLRs) by either shielding APOB and APOE or altering their conformation. Binding of chylomicrons and VLDL particles to lipolysis-stimulating receptors (LSRs) is also significantly inhibited by APOC3.

[0006] Elevated APOC3 levels lead to the development of hypertriglyceridemia, or high triglyceride blood concentrations. Elevated triglyceride levels are associated with a variety of diseases, including cardiovascular disease, atherosclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance), hypertension, and skin lesions (xanthomas). Extremely high triglyceride levels also increase the risk of acute pancreatitis. Therefore, regulating APOC3 metabolism may be an important new therapeutic approach for managing hypertriglyceridemia and related diseases. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, in this field of technology, there is a need for APOC3 expression regulators to treat apolipoprotein C3-related disorders such as hypertriglyceridemia. [Means for solving the problem]

[0008] This invention provides an iRNA composition that inhibits or reduces the expression of the APOC3 gene. This gene may be present in cells, for example, in cells within the body of a subject such as a human.

[0009] The present invention also provides methods and therapies for treating subjects having apolipoprotein C3-related disorders, such as hypertriglyceridemia, which may benefit from the inhibition or reduction of APOC3 gene expression, using iRNA compositions that inhibit or reduce APOC3 gene expression.

[0010] In some embodiments, the present invention provides a double-stranded RNAi agent for inhibiting the expression of apolipoprotein C3 (APOC3) in cells, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from the nucleotide sequence of SEQ ID NO: 2. Substantially all nucleotides in at least one strand are modified nucleotides, and the sense strand is coupled to a ligand attached to its 3' end.

[0011] In a particular embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides. In one embodiment, the sense strand and antisense strand include a complementary region comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the sequences listed in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13.

[0012] In some embodiments, at least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-fixed nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramido salt, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, or a 1,5- The group consists of anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing 5'-phosphate or a 5'-phosphate mimetic, nucleotides containing vinyl phosphate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to cholesteryl derivatives or dodecanoate bisdecylamide groups.

[0013] In one embodiment, substantially all of the nucleotides in the sense strand are modified. In another embodiment, substantially all of the nucleotides in the antisense strand are modified. In yet another embodiment, substantially all of the nucleotides in both the sense strand and the antisense strand are modified nucleotides. In one embodiment, all of the nucleotides in the sense strand are modified nucleotides. In another embodiment, all of the nucleotides in the antisense strand are modified nucleotides. In yet another embodiment, all of the nucleotides in both the sense strand and the antisense strand are modified nucleotides.

[0014] In one embodiment, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides.

[0015] In some embodiments, the present invention provides a double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a region complementary to a portion of the mRNA encoding APOC3, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3' Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5'(III) [In formula: i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; Each Na and Na' independently represents an oligonucleotide sequence containing 0–25 nucleotides, which are either modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; Each Nb and Nb' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is either modified, unmodified, or a combination thereof; Each np, np', nq, and nq' (each of which may or may not be present) independently represents an overhang 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 on Nb are different from the modifications on Y, and the modifications on Nb' are different from the modifications on Y'] as indicated by; and The sense chain is coupled to at least one ligand.

[0016] In a further 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 yet another further 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. In yet another embodiment, the YYY motif is located at or near the cleavage site of the sense strand. In yet another embodiment, the Y'Y'Y' motif is located at positions 11, 12 and 13 from the 5' end of the antisense strand.

[0017] In one embodiment, Y' is 2'-O-methyl or 2'-fluoro.

[0018] In some embodiments, equation (III) is equation (IIIa): Sense: 5'np-Na-YYY-Na-nq3' Antisense: 3'np'-Na'-Y'Y'Y'-Na'-nq'5'(IIIa) It is represented by [this].

[0019] In a further embodiment, the double-stranded region is 15 to 30 nucleotide pairs long. In another embodiment, the double-stranded region is 17 to 23 nucleotide pairs long. In yet another embodiment, the double-stranded region is 17 to 25 nucleotide pairs long. In yet another embodiment, the double-stranded region is 23 to 27 nucleotide pairs long. In yet another embodiment, the double-stranded region is 19 to 21 nucleotide pairs long. In yet another embodiment, the double-stranded region is 21 to 23 nucleotide pairs long.

[0020] In one embodiment, each chain has 15 to 30 nucleotides. In a further embodiment, each chain has 19 to 30 nucleotides.

[0021] In one aspect, the modification on the nucleotide is selected from the group consisting of modifications as listed in Tables 5, 9, 10, 11B, 12, 13, and combinations thereof.

[0022] In some embodiments, the modification on the nucleotide is 2'-O-methyl and 2'-fluoro modifications.

[0023] In some embodiments, the ligand is one or more GalNAc derivatives added via a divalent or trivalent branched linker. In further embodiments, the ligand is

Chemical formula

[0024] In some aspects, the ligand is added to the 3'-end of the sense strand.

[0025] In certain embodiments, the RNAi agent is conjugated with a ligand as shown in the following schematic diagram

Chemical formula

[0026] In some aspects, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate nucleotide internucleotide bond. In further aspects, the phosphorothioate or methylphosphonate nucleotide internucleotide bond is at the 3'-end of one strand. In another further aspect, this strand is the antisense strand. In yet another further aspect, this strand is the sense strand.

[0027] In some embodiments, the phosphorothioate or methylphosphonate nucleotide internucleotide bond is at the 5'-end of one strand. In further aspects, this strand is the antisense strand. In another further aspect, this strand is the sense strand.

[0028] <00In certain embodiments, the phosphorothioate or methylphosphonate internucleotide bond is located at both the 5' and 3' ends of one of the strands. In one embodiment, this strand is an antisense strand.

[0029] In some embodiments, the RNAi agent contains 6 to 8 phosphorothioate nucleotide interbonds. In further embodiments, the antisense strand contains 2 phosphorothioate nucleotide interbonds at its 5' end and 2 phosphorothioate nucleotide interbonds at its 3' end, and the sense strand contains at least 2 phosphorothioate nucleotide interbonds at either its 5' or 3' end.

[0030] In some embodiments, the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair.

[0031] In some embodiments, the Y nucleotide contains a 2'-fluoro modification. In further embodiments, the Y' nucleotide contains a 2'-O-methyl modification.

[0032] In some embodiments, the sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides.

[0033] In some embodiments, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, and 12.

[0034] In certain embodiments, the present invention also provides a double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, The sense strand contains 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 13), and the antisense strand contains 5'-AGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 14). Substantially all nucleotides in the sense strand and substantially all nucleotides in the antisense strand are modified nucleotides. The sense chain is coupled to the ligand attached to its 3' end, and The ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0035] In another embodiment, the present invention also provides a double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, The sense strand contains 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 13), and the antisense strand contains 5'-UGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 15), Substantially all nucleotides in the sense strand and substantially all nucleotides in the antisense strand are modified nucleotides. The sense chain is coupled to the ligand attached to its 3' end, and The ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0036] In certain embodiments, the present invention also provides a double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, The sense strand contains 5'-GCUUAAAAGGGACAGUAUUCA-3' (SEQ ID NO: 659), and the antisense strand contains 5'-UGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 670). Substantially all nucleotides in the sense strand and substantially all nucleotides in the antisense strand are modified nucleotides. The sense chain is coupled to the ligand attached to its 3' end, and The ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

[0037] In one embodiment, all nucleotides of the sense strand are modified nucleotides. In another embodiment, all nucleotides of the antisense strand are modified nucleotides. In yet another embodiment, all nucleotides of both the sense strand and the antisense strand are modified nucleotides.

[0038] In a further embodiment, at least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-fixed nucleotide, a restricted ethyl nucleotide, a debasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl (hydroxly) modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramido salt, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, or a 1,5- The group consists of anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing 5'-phosphate or a 5'-phosphate mimetic, nucleotides containing vinyl phosphate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to cholesteryl derivatives or dodecanoate bisdecylamide groups.

[0039] In one embodiment, the RNAi agent contains 10 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the RNAi agent contains 9 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the RNAi agent contains 8 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the RNAi agent contains 7 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the RNAi agent contains 6 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the RNAi agent contains 5 or fewer nucleotides containing 2'-fluoro modifications. In yet another embodiment, the sense strand contains 4 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the sense strand contains 4 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the sense strand contains 3 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the sense strand contains 2 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the antisense strand contains 6 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the antisense strand contains 5 or fewer nucleotides containing 2'-fluoro modifications. In another embodiment, the antisense chain contains four or fewer nucleotides containing 2'-fluoro modifications. In yet another embodiment, the antisense chain contains three or fewer nucleotides containing 2'-fluoro modifications. In yet another embodiment, the antisense chain contains two or fewer nucleotides containing 2'-fluoro modifications.

[0040] In one embodiment, the double-stranded RNAi agent of the present invention further comprises a 5'-phosphate or a 5'-phosphate mimetic in the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimetic in the 5' nucleotide of the antisense strand. In a specific embodiment, the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

[0041] In a particular embodiment, the ligand is [ka] That is the case.

[0042] In some embodiments, the RNAi agent is shown in the following schematic diagram. [ka] It is conjugated with a ligand as shown in [wherein X is O or S].

[0043] In some embodiments, the present invention provides a double-stranded RNAi agent comprising an RNAi sequence listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13.

[0044] In one embodiment, the RNAi agent has the following sequence: Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3'(Sequence ID 16) The antisense is AD-57553, which contains 5'asGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsAfsa 3' (sequence number 17).

[0045] In another embodiment, the RNAi agent has the following sequence: Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3'(Sequence ID 18) The antisense is AD-65696, which contains 5'VPusGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsasa 3' (SEQ ID NO: 19).

[0046] In yet another embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaAfaGfGfGfacaguauucaL96 3'(Sequence ID 20) The antisense is AD-65703, which contains 5'usGfsaauAfcUfGfucccUfuUfuaagcsasa 3' (Sequence ID 21).

[0047] In yet another embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaAfaGfGfGfacaguauucaL96 3'(Sequence ID 22) The antisense is AD-65704, which contains 5'usGfsaauacugucccUfuuuaagcsasa 3' (SEQ ID NO: 23).

[0048] In yet another embodiment, the RNAi agent has the following sequence: Sense: 5'cscscaauAfaAfGfCfuggacaagaaL96 3'(Sequence ID 714) The antisense is AD-67221, which contains 5'usUfscuuGfuCfCfagcuUfuAfuugggsasg 3' (sequence number 718).

[0049] In one embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaaaGfgGfacaguauuca 3' (Sequence ID 738) The antisense is AD-69535, which contains 5'sGfsaauacugucCfcUfuuuaagcsasa 3' (sequence number 749).

[0050] In another embodiment, the RNAi agent has the following sequence: Sense: 5'gscsuuaaaaGfgGfacagu(Agn)uuca 3'(Sequence ID 744) The antisense is AD-69541, which contains 5'usGfsaauacugucCfcUfuuuaagcsasa 3' (sequence number 755).

[0051] In certain embodiments, the present invention also provides compositions comprising a modified antisense polynucleotide agent having the ability to inhibit APOC3 expression in cells and comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, wherein the polynucleotide is approximately 14 to approximately 30 nucleotides long.

[0052] In some embodiments, the present invention also provides vectors containing double-stranded RNAi agents as described herein. In other embodiments, the present invention also provides cells containing double-stranded RNAi agents as described herein.

[0053] In some embodiments, the present invention relates to a composition comprising a double-stranded RNAi agent or a modified antisense polynucleotide agent as described herein, or a pharmaceutical composition comprising a vector.

[0054] In certain embodiments, the double-stranded RNAi agent is present in a non-buffer. In further embodiments, the non-buffer is physiological saline or water. In other embodiments, the double-stranded RNAi agent is present in a buffer. In further embodiments, the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In specific embodiments, the buffer is phosphate-buffered saline (PBS).

[0055] In one embodiment, the present invention also provides a method for inhibiting apolipoprotein C3 (APOC3) expression in cells, the method being: (a) The step of contacting cells with a composition, vector, or pharmaceutical composition comprising a double-stranded RNAi agent or a modified antisense polynucleotide agent as described herein, (b) The step of maintaining the cells produced in step (a) for a sufficient amount of time to achieve degradation of the mRNA transcript of the APOC3 gene, thereby inhibiting the expression of the APOC3 gene in the cells.

[0056] In one embodiment, the cells are present in the body of the subject. In a further embodiment, the subject is human or rabbit. In one embodiment, the subject suffers from APOC3-related disease.

[0057] In some embodiments, APOC3 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0058] In some embodiments, the present invention provides a method for treating a subject having an apolipoprotein C3 (APOC3) related disease, the method comprising administering a therapeutically effective amount of a composition or vector or pharmaceutical composition comprising a double-stranded RNAi agent or a modified antisense polynucleotide agent as described herein to the subject, thereby treating the subject.

[0059] In one embodiment, APOC3-related disease is hypertriglyceridemia. In another embodiment, APOC3-related disease is selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance), hypertension, atherosclerosis, and pancreatitis.

[0060] In some embodiments, the double-stranded RNAi agent is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg or approximately 0.5 mg / kg to approximately 50 mg / kg. In further embodiments, the double-stranded RNAi agent is administered in doses of approximately 10 mg / kg to approximately 30 mg / kg. In another embodiment, the double-stranded RNAi agent is administered in doses of approximately 3 mg / kg. In yet another embodiment, the double-stranded RNAi agent is administered in doses of approximately 10 mg / kg.

[0061] In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In another embodiment, the double-stranded RNAi agent is administered intravenously. In yet another embodiment, the double-stranded RNAi agent is administered intramuscularly.

[0062] In some embodiments, RNAi agents are administered in two or more doses. In further embodiments, RNAi agents are administered at intervals selected from the group consisting of approximately once every 12 hours, approximately once every 24 hours, approximately once every 48 hours, approximately once every 72 hours, and approximately once every 96 hours.

[0063] In certain embodiments, the method of the present invention further comprises the step of administering an additional therapeutic agent to a subject. In further embodiments, the additional therapeutic agent is selected from the group consisting of HMG-CoA reductase inhibitors, fibrates, bile acid scavengers, niacin, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol esterification protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTTP) inhibitors, cholesterol modulators, bile acid modulators, peroxisome proliferation-activating receptor (PPAR) agonists, gene-based therapies, complex vasoprotective agents, glycoprotein Ilb / IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors, squalene synthase inhibitors, monocyte chemotactic protein (MCP)-I inhibitors, or fish oil. [Brief explanation of the drawing]

[0064] [Figure 1] This bar graph shows the relative amount of APOC3 mRNA in Hep3B cells after treatment with a single dose of 0.1 nM or 10 mM of the iRNA of the present invention as indicated. [Figure 2] This bar graph shows the relative levels of APOC3 mRNA measured on day 5 in wild-type mice treated with GalNac-conjugated AD-57558 at doses of 3, 10, and 30 mg / kg. [Figure 3] This bar graph shows the measured APOC3 mRNA levels in individual APOC3-AAV mice injected with AD-57553, AD-57547, and AD-58924. [Figure 4] This bar graph shows the group-average APOC3 mRNA levels measured in APOC3-AAV mice injected with AD-57553, AD-57547, and AD-58924. [Figure 5]This bar graph shows the relative amounts of APOC3 mRNA measured in APOC3-AAV mice that were pre-injected with 1011 hAPOC3 AAV genome copies, followed by injection with AD-57553 at doses of 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg. [Figure 6] This bar graph shows the group mean relative amounts of APOC3 mRNA measured in APOC3-AAV mice that were pre-injected with 1011 hAPOC3 AAV genome copies, followed by injection with AD-57553 at doses of 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg. [Figure 7A] This graph shows the time course of serum APOC3 protein over 20 days in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection with the iRNA of the present invention at a dose of 3 mg / kg as instructed. [Figure 7B] This graph shows the time course of serum APOC3 protein over 30 days in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection with the iRNA of the present invention at a dose of 3 mg / kg as instructed. [Figure 8] This bar graph shows the amount of serum APOC3 protein measured on day 10 in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection with the iRNA of the present invention at a dose of 3 mg / kg as instructed. [Figure 9] This bar graph shows the amount of serum APOC3 protein measured on day 20 in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection with the iRNA of the present invention at a dose of 3 mg / kg as instructed. [Figure 10] This shows the time course of APOC3 protein levels measured in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection with the iRNA of the present invention at a dose of 3 mg / kg. [Figure 11]This is a schematic diagram showing the medication schedule Q2W×4 used in the multi-dose trials with AD-57553, AD-65696, AD-65699, AD-65703, and AD-65704. [Figure 12] Figure 12A shows the time course of APOC3 protein levels measured in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by four injections of the iRNA of the present invention at a dose of 0.3 mg / kg according to the dosing schedule shown in Figure 11. Figure 12B shows the time course of APOC3 protein levels measured in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by four injections of the iRNA of the present invention at a dose of 1 mg / kg according to the dosing schedule shown in Figure 11. Figure 12C shows the time course of APOC3 protein levels measured in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by four injections of the iRNA of the present invention at a dose of 3 mg / kg according to the dosing schedule shown in Figure 11. [Figure 13] This bar graph shows the relative levels of APOC3 protein measured on day 14 in APOC3-AAV mice that were injected with 1011 hAPOC3 AAV genome copies, followed by single doses of AD-65704 at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. [Figure 14] This bar graph shows the relative amount of APOC3 protein measured 20 days after the final dose in APOC3-AAV mice that were injected with 1011 hAPOC3 AAV genome copies, followed by multiple doses of AD-65704 at 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, according to the drug administration schedule shown in Figure 11. [Figure 15] This shows the time course of APOC3 protein levels measured in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection with the iRNA of the present invention at a dose of 1 mg / kg. [Figure 16]Figure 16A is a bar graph showing the relative amount of APOC3 protein measured on day 10 in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection of the iRNA of the present invention at a single dose of 1 mg / kg. Figure 16B is a bar graph showing the relative amount of APOC3 protein measured on day 24 in APOC3-AAV mice that were injected with 10¹¹ hAPOC3 AAV genome copies, followed by injection of the iRNA of the present invention at a single dose of 1 mg / kg. [Figure 17] Figure 17A is a bar graph showing the relative amount of serum APOC3 protein measured on day 14 in APOC3-AAV mice injected with 10¹¹ hAPOC3 AAV genome copies, followed by a single dose of 1 mg / kg of indicated iRNA. Figure 17B is a graph showing the amount of serum APOC3 protein at days 0, 14, 28, and 42 relative to pre-administration levels in APOC3-AAV mice injected with 10¹¹ hAPOC3 AAV genome copies, followed by a single dose of 1 mg / kg of indicated iRNA. [Figure 18] Figure 18A is a graph showing the amount of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, 36, 43, 57, 64, and 71 in cynomolgus monkeys after a single dose of 1 mg / kg of AD-65704 over 8 weeks, compared to pre-administration levels on day 7. Figure 18B is a graph showing the amount of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, and 36 in cynomolgus monkeys after a single dose of 1 mg / kg of AD-65704, compared to pre-administration levels on day 7. Figure 18C is a graph showing the amount of liver APOC3 mRNA in cynomolgus monkeys on day 64 after a single dose of AD-65704 at a weekly dose of 1 mg / kg (q1w × 5) over 5 weeks, compared to the amount before administration on day -7, and the amount of liver APOC3 mRNA in cynomolgus monkeys on day 12 after a single dose of AD-65704 at a weekly dose of 1 mg / kg, compared to the amount before administration on day -7. [Figure 19]Figure 19A is a graph showing the amount of serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, and 36 in cynomolgus monkeys after a single dose of 1 mg / kg of the indicated iRNA, compared to the pre-administration level on day 7. Figure 19B is a bar graph showing the amount of liver APOC3 mRNA measured on day 12 in cynomolgus monkeys after a single dose of 1 mg / kg of the indicated iRNA, compared to the pre-administration level on day 7. [Figure 20] Figure 20A is a graph showing the amount of serum APOC3 mRNA measured on days 1, 8, 11, 15, 22, 29, 36, 43, 50, 57, 64, and 71 in cynomolgus monkeys after a single dose of 1 mg / kg of the indicated iRNA and a subsequent single subcutaneous dose of 3 mg / kg of the same drug on day 36, compared to pre-administration levels on day -7. Figure 20B is a bar graph showing the amount of liver APOC3 mRNA measured on day 12 in cynomolgus monkeys after a single dose of 1 mg / kg of the indicated iRNA on day 1 and a subsequent single dose of 3 mg / kg of the same iRNA agent on day 36, compared to pre-administration levels on day -7. [Modes for carrying out the invention]

[0065] The present invention provides iRNA agents, such as double-stranded iRNA agents, and compositions that reduce or inhibit the expression of the APOC3 gene. This gene may be present in cells, for example, in cells within the body of an object such as a human.

[0066] The present invention also provides a method for treating subjects with apolipoprotein C3-related diseases or disorders, such as hypertriglyceridemia, which may benefit from inhibition or reduction of APOC3 expression, using an iRNA composition that inhibits or reduces the expression of the APOC3 gene.

[0067] The iRNAs of the present invention are approximately 30 nucleotides or less in length, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19- The iRNAs include RNA strands (antisense strands) having regions of 24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide lengths, where these regions are substantially complementary to at least a portion of the mRNA transcript of the APOC3 gene. These iRNAs enable the targeted degradation of APOC3 gene mRNA in cells. Specifically, extremely low doses of the iRNAs of the present invention can specifically and efficiently mediate RNA interference (RNAi), potentially leading to significant inhibition of APOC3 gene expression. The inventors have demonstrated, using in vitro and in vivo assays, that iRNAs targeting the APOC3 gene can mediate RNAi, resulting in significant inhibition of APOC3 gene expression and a decrease in APOC3 protein levels. They have also demonstrated that iRNAs targeting the APOC3 gene can reduce symptoms associated with apolipoprotein C3-related disorders, such as lowering triglyceride levels. Therefore, methods and compositions containing these iRNAs are useful for treating subjects with apolipoprotein C3-related disorders (disoerders), such as hypertriglyceridemia.

[0068] The following detailed description discloses how to prepare and use compositions containing iRNA to inhibit APOC3 gene expression, as well as compositions, uses, and methods for treating subjects with diseases and disorders that may benefit from the inhibition and / or reduction of APOC3 expression.

[0069] I. Definition To make the present invention easier to understand, certain terms are defined first. In addition, it should be noted that whenever parameter values ​​or ranges of values ​​are enumerated, intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of the present invention.

[0070] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical purposes of the article. For example, "an element" means one factor or two or more factors, such as multiple factors.

[0071] The term "including" is used herein to mean "including, but not limited to," and is used without distinction from that term.

[0072] In this specification, the term "or" is used to mean, and is used without distinction from, the term "and / or" unless the context clearly indicates otherwise.

[0073] As used herein, the term "APOC3" refers to the well-known gene encoding apolipoprotein C3, as well as its protein product, also known in the art as HALP2 or APOCIII.

[0074] The term "APOC3" can refer to: human APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:4557322 (NM_000040.1; SEQ ID NO: 1)); Macaca fascicularis APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:544489959 (XM_05579730.1, SEQ ID NO: 3)); Macaca mulatta APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:297269260 (XM_001090312.2; SEQ ID NO: 5)); and mouse (Mus musculus (Mus This includes musculus APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:577019555 (NM_023114.4, SEQ ID NO: 7)); rat (Rattus norvegicus) APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank accession number GI:402534545 (NM_012501.2, SEQ ID NO: 9)); and rabbit (Oryctolagus cuniculus), GenBank accession number GI:655601498 (XM_002708371.2, SEQ ID NO: 11).

[0075] Further examples of APOC3 mRNA sequences are readily available in publicly accessible databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0076] When used herein, the term "APOC3" also refers to naturally occurring DNA sequence variations of the APOC3 gene, such as single nucleotide polymorphisms (SNPs) in the APOC3 gene. Exemplary SNPs of the APOC3 DNA sequence can be referenced in the dbSNP database available at www.ncbi.nlm.nih.gov / projects / SNP / . Non-exclusive examples of sequence variations within the APOC3 gene include, for example, two mutations rs2854116 and rs2854117, described in Petersen, KFet al., (2010), N.Engl.J.Med.362(12):1082-1089 (all of which are incorporated herein by reference).

[0077] In the use of this specification, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the APOC3 gene, including mRNA, which is the RNA processing product of the primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage, either in or near the portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the APOC3 gene.

[0078] The target sequence may be approximately 9 to 36 nucleotides long, for example, approximately 15 to 30 nucleotides long. For example, the target sequences may be 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-2 6, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, etc., which may be approximately 15-30 nucleotide lengths. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0079] In the use of this specification, the term “sequence-containing chain” refers to an oligonucleotide containing a nucleotide chain described by the sequence referred to, using standard nucleotide nomenclature.

[0080] "G," "C," "A," "T," and "U" typically represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, the terms "ribonucleotide" or "nucleotide" are also understood to refer to modified nucleotides or substituted portions, as will be further detailed below (see, for example, Table 3). Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be replaced with other portions without substantially altering the base-pairing properties of oligonucleotides containing such substituted portions. As an example not intended to be limiting, a nucleotide containing inosine as a base can base-pair with adenine, cytosine, or uracil-containing nucleotides. Thus, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the dsRNAs discussed in this invention with, for example, nucleotides containing inosine. In another embodiment, adenine and cytosine may be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form GU fluctuation base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods discussed in this invention.

[0081] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” are used synonymously herein and refer to agents containing RNA as defined herein that mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNAs induce sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate, for example, inhibit APOC3 expression in cells, such as those in mammalian subjects.

[0082] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as the APOC3 target mRNA sequence, and induces cleavage of the target RNA. Although we do not wish to be constrained by theory, it is thought that long double-stranded RNA introduced into cells is degraded into double-stranded small interfering RNA (siRNA) containing sense and antisense strands by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). When Dicer, a ribonuclease III-like enzyme, processes these dsRNAs, they become small interfering RNAs of 19-23 base pairs with a characteristic 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing a complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one embodiment, the present invention relates to a single-stranded RNA (ssRNA) (antisense strand of an siRNA double helix) that is generated in a cell and promotes the formation of the RISC complex, resulting in the silencing of a target gene, namely the APOC3 gene. Accordingly, the term "siRNA" also refers to RNAi as described above.

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

[0084] In another embodiment, the “iRNA” used in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” “RNAi agent,” “RNAi,” or “dsRNA.” The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are referred to as having “sense” and “antisense” directions with respect to the target RNA, i.e., the APOC3 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces degradation of target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0085] Generally, the majority of nucleotides in each strand of a 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. In addition, as used herein, “RNAi agent” may include ribonucleotides having chemical modifications; an RNAi agent may include substantial modifications to multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide independently having a modified sugar moiety, a modified nucleotide-nucleotide bond, and / or a modified nucleic acid base. Thus, the term modified nucleotide includes substitution, addition, or removal of, for example, functional groups or atoms to nucleoside bonds, sugar moieties, or nucleic acid bases. Modifications suitable for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. Any such modification, when used in an siRNA-type molecule, is included in “RNAi agent” for the purposes of this specification and the claims.

[0086] The double-stranded region may be of any length that enables the specific degradation of the desired target RNA via the RISC pathway, and may be in the range of approximately 9 to 36 base pairs in length, for example, approximately 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example, approximately 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, These include base pair lengths of 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0087] The two strands forming the double-stranded structure may be different parts of a larger RNA molecule, or they may be different RNA molecules. If the two strands are parts of one larger molecule, and thus the 3' end of one strand forming the double-stranded structure is joined to the 5' end of the other strand by an uninterrupted nucleotide strand, the joined RNA strands are referred to as a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide; in some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, and at least 23 or more unpaired nucleotides.

[0088] If two substantially complementary strands of dsRNA are composed of another RNA molecule, these molecules may, but do not necessarily, be covalently linked. If the two strands are covalently linked between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand by means other than an uninterrupted nucleotide chain, the linking structure is called a "linker." RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs.

[0089] In one embodiment, the RNAi agent of the present invention is a dsRNA, each strand containing 20-30 nucleotides that interact with a target RNA sequence, such as the APOC3 target mRNA sequence, and induces cleavage of the target RNA. Although we do not wish to be constrained by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). When Dicer, a ribonuclease III-like enzyme, processes dsRNA, it becomes a small interfering RNA of 19-23 base pairs with a characteristic 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0090] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of dsRNA extends over the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang may contain, or consist of, nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, the 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0091] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, which overhang at the 3' and / or 5' ends. In another embodiment, one or more nucleotides in the overhang are substituted with thiophosphate nucleosides.

[0092] "Blunt-ended" or "blunt-ended" means that the end of a double-stranded RNAi agent has no unpaired nucleotides, i.e., no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at any end of the molecule. The RNAi agents of the present invention include RNAi agents having a nucleotide overhang at one end (i.e., an agent having one overhang and one blunt end) or RNAi agents having nucleotide overhangs at both ends.

[0093] The terms “antisense strand” or “guide strand” refer to an iRNA strand, such as dsRNA, that contains a region substantially complementary to a target sequence, such as APOC3 mRNA. As used herein, the term “regional complementarity” refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as the APOC3 nucleotide sequence as defined herein. If the complementary region is not perfectly complementary to the target sequence, there may be a mismatch in the internal or terminal regions of the molecule. Generally, the most tolerable mismatches are in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the iRNA.

[0094] The terms “sense strand” or “passenger strand” as used herein refer to an iRNA strand that includes a region substantially complementary to the antisense strand region as defined herein.

[0095] As used herein, the term “cleavage region” refers to a region located immediately adjacent to a cleavage site. A cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region includes three bases on both sides of the cleavage site and immediately adjacent thereto. In some embodiments, the cleavage region includes two bases on both sides of the cleavage site and immediately adjacent thereto. In some embodiments, the cleavage site is specifically located at the binding sites of nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0096] In the use of this specification, unless otherwise specified, the term “complementary” refers to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under specific conditions to form a double-stranded structure, as will be understood by those skilled in the art when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically reasonable conditions that may be encountered in living organisms, may also be applicable. Those skilled in the art can determine the optimal set of conditions for the complementarity test of the two sequences, depending on the end use of the hybridized nucleotides.

[0097] For example, complementary sequences within iRNAs, such as those within dsRNAs, as described herein, include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence, spanning the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred to herein as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, generally 5, 4, 3, or 2 or fewer, while retaining the ability to hybridize into double-stranded hybridization of up to 30 base pairs under conditions most appropriate for their end use, such as inhibition of gene expression via the RISC pathway. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, where the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, is still referred to as “perfectly complementary” for the purposes described herein.

[0098] "Complementary" sequences, as used herein, also include, or may be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, insofar as the above requirements regarding their hybridizing ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairs or Hoogsteen-type base pairs.

[0099] In this specification, the terms “complementary,” “fully complementary,” and “substantially complementary” may be used in relation to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand and target sequence of an iRNA agent, as will be understood from the context in which they are used.

[0100] As used herein, a polynucleotide "substantially complementary to at least a portion" of messenger RNA (mRNA) means a polynucleotide substantially complementary to the continuous portion of the mRNA in question (e.g., the mRNA encoding APOC3). For example, a polynucleotide is complementary to at least a portion of APOC3 mRNA if its sequence is substantially complementary to the uninterrupted portion of the mRNA encoding APOC3.

[0101] Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target APOC3 sequence. In other embodiments, the sense strand polynucleotides and / or antisense polynucleotides disclosed herein are substantially complementary to the target APOC3 sequence and comprise a continuous nucleotide sequence that is at least about 80% complementary over an equivalent region of any one of the nucleotide sequences of SEQ ID NOs. 1 to 12 and its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary, or a fragment of any one of the SEQ ID NOs. 1 to 12.

[0102] In one embodiment, the RNAi agent of the present invention comprises an antisense polynucleotide complementary to a target APOC3 sequence and a sense strand substantially complementary thereto, wherein the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary over an equivalent region of any one of the nucleotide sequences of SEQ ID NOs: 1 to 12 and its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary, or a fragment of any one of the SEQ ID NOs: 1 to 12. In another embodiment, the RNAi agent of the present invention comprises an antisense strand substantially complementary to the target APOC3 sequence, comprising a continuous nucleotide sequence that is at least about 80% complementary over the equivalent region of any one of the nucleotide sequences of SEQ ID NOs: 1 to 12 and its entire length, for example, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary, or a fragment of any one of the SEQ ID NOs: 1 to 12.

[0103] Generally, the majority of nucleotides in each chain are ribonucleotides, but as described in detail herein, one or both chains may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, “iRNA” can refer to chemically modified ribonucleotides. Such modifications include all types of modifications disclosed herein or known in the art. For the purposes of this specification and the claims, any such modification is encompassed by “iRNA” in its use in an iRNA molecule.

[0104] In one aspect of the present invention, the agent used in the methods and compositions of the present invention is a single-stranded antisense nucleic acid molecule that inhibits target mRNA by an antisense inhibition mechanism. The single-stranded antisense nucleic acid molecule is complementary to a sequence in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by physically interfering with base pairing and translation mechanisms with mRNA (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense nucleic acid molecule may be about 15 to about 30 nucleotides long and may have a sequence complementary to the target sequence. For example, the single-stranded antisense nucleic acid molecule may contain a sequence that is at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0105] In the use of this specification, “Subject” means a mammal, including primates (such as humans, non-human primates such as monkeys and chimpanzees), non-primates (such as cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or an animal such as a bird (such as a duck or goose). In one embodiment, the subject is a human being treated or evaluated for a disease, disorder or condition that would benefit from reduced APOC3 expression; a human being at risk of a disease, disorder or condition that would benefit from reduced APOC3 expression; a human being having a disease, disorder or condition that would benefit from reduced APOC3 expression; and / or a human being treated for a disease, disorder or condition that would benefit from reduced APOC3 expression as described herein.

[0106] When used herein, the terms “treat” or “treat” mean beneficial or desired outcomes, including, but not limited to, reduction or improvement, of one or more symptoms associated with undesirable or excessive APOC3 expression, such as hypertriglyceridemia (or high triglyceride levels). Such symptoms may include, for example, skin symptoms (e.g., xanthomas); eye abnormalities (e.g., retinal dyslipidemia); hepatosplenomegaly (enlargement of the liver and spleen); neurological symptoms; or abdominal pain attacks, which may be mild episodes of pancreatitis. Other symptoms associated with undesirable or excessive APOC3 expression may also include any symptoms of diseases, disorders or conditions that may be caused by, associated with, or resulting from hypertriglyceridemia, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis. "Treatment" can also mean an extension of survival compared to the survival expected without treatment.

[0107] In the context of the APOC3 level or disease marker or symptom in question, the term “lower” refers to a statistically significant decrease in such level. This decrease may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher, preferably down to a level recognized as within the normal range for individuals without such disorder.

[0108] As used herein, “prevention” or “prevention” means, when used in relation to a disease, disorder, or condition that can be treated or improved by reducing the expression of the APOC3 gene, a reduction in the likelihood of developing symptoms associated with such disease, disorder, or condition, such as symptoms of undesirable or excessive APOC3 expression, such as hypertriglyceridemia. For example, the likelihood of developing hypertriglyceridemia is reduced if, for example, an individual with one or more risk factors for hypertriglyceridemia does not develop hypertriglyceridemia, or develops hypertriglyceridemia with lower severity compared to a population with the same risk factors but not receiving treatment as described herein. Not developing a disease, disorder, or condition, or a reduction in the development of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically recognized scale for the disease or disorder), or a delay in symptoms (e.g., by days, weeks, months, or years) is considered effective prevention.

[0109] As used herein, the term “apolipoprotein C3-related disorder” or “APOC3-related disorder” refers to a disease, disorder, or condition caused by or associated with undesirable or excessive APOC3 expression. The term “APOC3-related disorder” includes diseases, disorders, or conditions that can be treated or improved by reducing APOC3 expression. The term “APOC3-related disorder” also includes hypertriglyceridemia or high triglyceride levels.

[0110] The serum triglyceride levels in subjects that may show signs of hypertriglyceridemia, such as human subjects, are described in Oh, RC et al., (2007) American Family Physician, 75(9):1366-1371. Specifically, hypertriglyceridemia may be associated with “borderline hypertriglyceride levels” (i.e., 150–199 mg / dL or 1.70–2.25 mmol / L); “hypertriglyceride levels” (i.e., 200–499 mg / dL or 2.26–5.64 mmol / L); or “very hypertriglyceride levels” (i.e., 500 mg / dL or higher (or 5.65 mmol / L or higher)).

[0111] In one embodiment, APOC3-related disorders are primary triglyceridemias. Primary triglyceridemias are caused by environmental or genetic factors (e.g., as a result of an apparent underlying disease). Exemplary disorders characterized as primary triglyceridemias include, but are not limited to, familial chyleptomania (hyperlipoproteinemia type 1), primary mixed hyperlipidemia (type 5), familial hypertriglyceridemia (hyperlipoproteinemia type 4), familial combined hyperlipoproteinemia (type 2B), and familial anomalous β-lipoproteinemia (hyperlipoproteinemia type 3).

[0112] In another embodiment, APOC3-related disorder is secondary hypertriglyceridemia. "Secondary triglyceridemia" is caused by or associated with other underlying disorders and conditions. Such disorders and / or conditions include, for example, obesity, metabolic syndrome, diabetes mellitus, fatty liver, alcohol consumption, renal disease, pregnancy, non-alcoholic fatty liver disease, hypothyroidism, paraproteinemia (such as hypergammaglobulinemia in macroglobulinemia, myeloma, lymphoma and lymphocytic leukemia), autoimmune disorders (such as systemic lupus erythematosus), and medication use (such as antiretroviral drugs including ritonavir and lopinavir, and antipsychotic therapy including clozapine and olanzapine) (see G. Yuan et al., (2007) Canadian Medical Association Journal, 176(8):1113-1120).

[0113] Any disorder that may cause hypertriglyceridemia (e.g., secondary hypertriglyceridemia) or any disorder that may result from hypertriglyceridemia (e.g., primary or secondary hypertriglyceridemia) is included in the term "APOC3-related disorder." Non-limiting examples of APOC3-related disorders include metabolic disorders, e.g., non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance); hypertension; cardiovascular disorders, e.g., atherosclerosis; and pancreatitis, e.g., acute pancreatitis.

[0114] II. The iRNA of the present invention The present invention provides an iRNA that inhibits the expression of the APOC3 gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the APOC3 gene in cells, such as those in the body of mammals, such as humans, in subjects with APOC3-related diseases, such as hypertriglyceridemia. The dsRNA comprises an antisense strand having a complementary region complementary to at least a portion of the mRNA formed by APOC3 gene expression. The complementary region is approximately 30 nucleotides or less in length (for example, approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). When this iRNA comes into contact with cells expressing the APOC3 gene, it inhibits the expression of the APOC3 gene (e.g., human, primate, non-primate, or avian APOC3 gene) by at least approximately 10% when assayed by, for example, PCR or branched DNA (bDNA)-based methods, or by protein-based methods such as immunofluorescence analysis using, for example, Western blotting or flow cytometry.

[0115] A dsRNA contains two complementary RNA strands, which hybridize under the conditions in which the dsRNA is used to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary to the target sequence and generally fully complementary. The target sequence may originate from the mRNA sequence formed during the expression of the APOC3 gene. The other strand (the sense strand) contains a region complementary to the antisense strand such that, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere in this specification and as known in the art, the complementary sequence of a dsRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to that on a separate oligonucleotide.

[0116] Generally, double-stranded structures are, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 These range from 15 to 30 base pairs in length, such as 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths beyond the listed ranges and lengths are also intended to be part of the present invention.

[0117] Similarly, complementary regions of target sequences are, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-2 7, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide lengths, etc. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0118] In some embodiments, the dsRNA is about 15–20 nucleotides long, or about 25–30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. It is well known in the art that dsRNA longer than about 21–23 nucleotides may serve as a substrate for Dicer. As those skilled in the art will recognize, the target region of the RNA to be cleaved is in most cases part of a larger RNA molecule, which is often an mRNA molecule. Where applicable, the “part” of the mRNA target is a continuous sequence of mRNA targets long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0119] Those skilled in the art can, for example, use approximately 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-3 3, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-1 9, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20 It will also be recognized that double-stranded regions of approximately 9 to 36 base pairs, such as ~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29, 21~28, 21~27, 21~26, 21~25, 21~24, 21~23, or 21~22 base pairs, are the main functional parts of dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs, within the range that it is processed into a functional double-strand of 15 to 30 base pairs that targets a desired RNA for cleavage, is a dsRNA. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a native miRNA. In another embodiment, iRNA agents useful for targeting APOC3 expression are not generated in the target cell by cleaving larger dsRNAs.

[0120] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit surprisingly superior inhibitory properties compared to their blunt-end equivalents. Nucleotide overhangs may include, or consist of, nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. Overhangs may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0121] dsRNA can be synthesized by standard methods known in the art, using automated DNA synthesizers, such as those commercially available from Biosearch, Applied Biosystems, Inc., as will be discussed further below.

[0122] The iRNA compounds of the present invention may be prepared using a two-step method. First, the individual strands of a double-stranded RNA molecule are prepared separately. Next, the constituent strands are annealed. The individual strands of the siRNA compound can be prepared using solution phase, solid-phase organic synthesis, or both. Organic synthesis offers the advantage of easily preparing oligonucleotide chains containing non-natural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using solution phase, solid-phase organic synthesis, or both.

[0123] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand and the corresponding antisense strand are each selected from the group of sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. In this embodiment, one of these two sequences is complementary to the other of these two sequences, and one of these sequences is substantially complementary to the mRNA sequence resulting from APOC3 gene expression. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is described as the sense strand in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. In one embodiment, a substantially complementary sequence of dsRNA is contained in a separate oligonucleotide. In another embodiment, a substantially complementary sequence of dsRNA is contained in a single oligonucleotide.

[0124] While some of the sequences in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 are described as modified and / or conjugated sequences, it will be understood that the RNA of the iRNA of the present invention, for example, the dsRNA of the present invention, may include any one of the sequences shown in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, which are unmodified, unconjugated, and / or modified and / or conjugated in a manner different from those described therein.

[0125] Those skilled in the art are well aware that dsRNAs having double-stranded structures of approximately 20–23 base pairs, such as 21 base pairs, are supported as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures can also be equally effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, due to the nature of the oligonucleotide sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, the dsRNAs described herein may comprise at least one strand of a minimum length of 21 nucleotides. It can be reasonably predicted that shorter double-stranded sequences having one of the sequences from Table 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, with only a few nucleotides missing from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having at least 15, 16, 17, 18, 19, or 20 or more consecutive nucleotide sequences derived from one of the sequences from Table 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, and having the ability to inhibit APOC3 gene expression, differing from dsRNAs containing full-length sequences by approximately 5, 10, 15, 20, 25, or 30% or less, are intended to be within the scope of the present invention.

[0126] Furthermore, the RNAs provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 identify sites in the APOC3 transcript that are highly sensitive to RISC-mediated cleavage. Thus, the present invention further features iRNAs that target within one of these sequences. In the use herein, an iRNA is said to target within that specific site of the RNA transcript if it promotes cleavage of the transcript somewhere within that site. Such iRNAs generally consist of about 15 consecutive nucleotides from one of the sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, which are linked to an additional nucleotide sequence from a region adjacent to a selected sequence in the APOC3 gene.

[0127] Target sequences are generally about 15–30 nucleotides long, but there is a wide range of variation in the suitability of specific sequences within this range to induce cleavage of any given target RNA. The various software packages and guidelines presented herein provide guidance for identifying the optimal target sequence for any given gene target, but an empirical approach can also be taken to identify sequences within a size range that could act as the target sequence by actually or figuratively (including, for example, by computer simulation) placing a “window” or “mask” of a given size (21 nucleotides as an unrestricted example) on the target RNA sequence. By sequentially moving the sequence “window” one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing to identify optimally functioning sequences (using assays described herein or known in the art), can identify the RNA sequence that best mediates the inhibition of target gene expression when targeted with an iRNA agent. Therefore, while a sequence identified in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 represents an effective target sequence, it is possible to further optimize inhibition efficiency by identifying sequences with equivalent or better inhibitory properties by sequentially "walking a window" one nucleotide upstream or downstream of a given sequence.

[0128] Furthermore, it is explored that further optimization of any sequence identified in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 may be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking a window of size longer or shorter than the target RNA from that position. Again, combining this approach to creating new target candidates with testing the efficacy of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein may lead to further improvements in inhibition efficiency. Moreover, such optimized sequences may be modulated by further optimizing the molecule as an expression inhibitor (e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, targeting of specific sites or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes, etc.) by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or considered herein.

[0129] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA drug chain complementary to the APOC3 gene region, the RNA chain generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it may be determined whether an iRNA containing a mismatch with the target sequence is effective in inhibiting APOC3 gene expression. Examining the effectiveness of mismatched iRNAs in inhibiting APOC3 gene expression is important, especially when a particular complementary region of the APOC3 gene is known to have polymorphic sequence variations within a population.

[0130] III. Modified iRNA of the Invention In one embodiment, the RNA of the iRNA of the present invention, such as dsRNA, is undenatured and free from chemical modifications and / or bindings, such as those known in the art and described herein. In another embodiment, the RNA of the iRNA of the invention, such as dsRNA, is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all of the nucleotides of the iRNA of the present invention are modified nucleotides. The iRNA of the present invention that is "substantially all of its nucleotides are modified" is mostly modified, but not all, and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0131] In some embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified, and the iRNA agent contains 10 or fewer nucleotides containing 2'-fluoro modifications (for example, including 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). For example, in some embodiments, the sense strand contains 4 or fewer nucleotides containing 2'-fluoro modifications (for example, including 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other embodiments, the antisense strand contains 6 or fewer nucleotides containing 2'-fluoro modifications (for example, including 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In another aspect of the present invention, all nucleotides of the iRNA of the present invention are modified, and the iRNA agent contains 10 or fewer nucleotides including 2'-fluoro modifications (for example, including 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications).

[0132] Nucleic acids discussed herein can be synthesized and / or modified by methods established in the art, such as those described herein by reference in “Current protocols in nucleic acid chemistry,” Beaucage, S. Let al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Examples of modifications include terminal modifications such as 5'-end modifications (phosphorylation, conjugation, inversion) or 3'-end modifications (conjugation, DNA nucleotide, inversion); base modifications such as substitution, base removal (debasing nucleotide), or conjugated bases, whether at a stabilizing base, a destabilizing base, or a base that forms a base pair with an expanding partner repertoire; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and main chain modifications, including modification or substitution of phosphate diester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNA containing a modified main chain or RNA without natural nucleoside interbonding. RNAs having a modified backbone include those that do not have a phosphorus atom in their backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone are also considered oligonucleosides. In some embodiments, modified iRNAs have a phosphorus atom in their internucleoside backbone.

[0133] Examples of modified RNA backbone include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylenephosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages and their 2'-5' linked analogues, as well as boranophosphates with reversed polarity where adjacent nucleoside unit pairs are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0134] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above are U.S. Patent No. 3,687,808; U.S. Patent No. 4,469,863; U.S. Patent No. 4,476,301; U.S. Patent No. 5,023,243; U.S. Patent No. 5,177,195; U.S. Patent No. 5,188,897; U.S. Patent No. 5,264,423; U.S. Patent No. 5,276,019; U.S. Patent No. 5,278,302; U.S. Patent No. 5,286,7 Specification No. 17; US Patent Nos. 5,321,131; US ​​Patent Nos. 5,399,676; US Patent Nos. 5,405,939; US Patent Nos. 5,453,496; US Patent Nos. 5,455,233; US Patent Nos. 5,466,677; US Patent Nos. 5,476,925; US Patent Nos. 5,519,126; US Patent Nos. 5,536,821; US ​​Patent Nos. 5,541,316; US Patent Nos. 5,550,111; US ​​Patent Nos. 5,563,253; US Patent Nos. 5,57 U.S. Patent No. 1,799; U.S. Patent No. 5,587,361; U.S. Patent No. 5,625,050; U.S. Patent No. 6,028,188; U.S. Patent No. 6,124,445; U.S. Patent No. 6,160,109; U.S. Patent No. 6,169,170; U.S. Patent No. 6,172,209; U.S. Patent No. 6,239,265; U.S. Patent No. 6,277,603; U.S. Patent No. 6,326,199; U.S. Patent No. 6,346,614; U.S. Patent No. 6,444,423; U.S. Patent No. 6 U.S. Patent Nos. 531,590; U.S. Patent Nos. 6,534,639; U.S. Patent Nos. 6,608,035; U.S. Patent Nos. 6,683,167; U.S. Patent Nos. 6,858,715; U.S. Patent Nos. 6,867,294; U.S. Patent Nos. 6,878,805; U.S. Patent Nos. 7,015,315; U.S. Patent Nos. 7,041,816; U.S. Patent Nos. 7,273,933; U.S. Patent Nos. 7,321,029; and U.S. Patent No. RE39464, but not limited to these.

[0135] Modified RNA backchains that do not contain a phosphorus atom have backchains formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar portion of nucleosides); siloxane backchains; sulfide, sulfoxide, and sulfone backchains; formacetyl and thioformacetyl backchains; methyleneformacetyl and thioformacetyl backchains; alkene-containing backchains; sulfamate backchains; methyleneimino and methylenehydrazino backchains; sulfonate and sulfonamide backchains; those having amide backchains; and others having mixed N, O, S, and CH2 components.

[0136] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides are U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967, all of which are incorporated herein by reference in their entirety. Examples of U.S. Patent Nos. include, but are not limited to, U.S. Patent Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439.

[0137] In another embodiment, a suitable RNA mimetic is considered for use in iRNA, in which both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with a new group. The base units are maintained for hybridization with a suitable nucleic acid target compound. Such an oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bind directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, each of which is incorporated herein by reference in its entirety. Furthermore, PNA compounds suitable for use in iRNA according to the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0138] Some embodiments of the present invention include RNA having a phosphorothioate backbone, and oligonucleosides having a heteroatom backbone which is --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [natural phosphate diester backbone is represented as --O--P--O--CH2--], and an amide backbone as described in U.S. Patent No. 5,602,240. In some embodiments, the RNA discussed herein has the morpholino backbone structure described in the aforementioned U.S. Patent No. 5,034,506.

[0139] Modified RNA may also contain one or more substituted sugar moieties. For example, iRNAs such as the dsRNA discussed herein may contain at the 2' position one of OH;F;O-, S-, or N-alkyl;O-, S-, or N-alkenyl;O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2 (wherein n and m are 1 to about 10). In another embodiment, the dsRNA contains one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intervener, group that improves the pharmacokinetic properties of iRNA, or group that improves the pharmacodynamic properties of iRNA, and other substituents having similar properties. In some embodiments, the modification contains 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications are 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described below in the examples herein.

[0140] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the iRNA, specifically at the 3' position of the sugar on the 3' terminal nucleotide, or in the 2'-5' linked dsRNA, and at the 5' position of the 5' terminal nucleotide. The iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of the above-mentioned modified sugar structures include, specifically, U.S. Patent No. 4,981,957; U.S. Patent No. 5,118,800; U.S. Patent No. 5,319,080; U.S. Patent No. 5,359,044; U.S. Patent No. 5,393,878; U.S. Patent No. 5,446,137; U.S. Patent No. 5,466,786; U.S. Patent No. 5,514,785; U.S. Patent No. 5,519,134; U.S. Patent No. 5, U.S. Patent Nos. 567,811; U.S. Patent Nos. 5,576,427; U.S. Patent Nos. 5,591,722; U.S. Patent Nos. 5,597,909; U.S. Patent Nos. 5,610,300; U.S. Patent Nos. 5,627,053; U.S. Patent Nos. 5,639,873; U.S. Patent Nos. 5,646,265; U.S. Patent Nos. 5,658,873; U.S. Patent Nos. 5,670,633; and U.S. Patent Nos. 5,700,920, among others. The entire contents of each of the above are incorporated herein by reference.

[0141] iRNAs may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). In the use of this specification, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include deoxythymine (dT), 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; and 5-uracil (pseudouracil). Other examples of synthetic and natural nucleic acid bases include 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-daazaadenine; and 3-deazaguanine and 3-deazaadenine.Furthermore, examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, STand Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276–278), making it an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0142] Representative U.S. patents teaching the preparation of the specific modified nucleic acid bases and other modified nucleic acid bases described above are, as their entire contents are incorporated herein by reference, U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469. U.S. Patent Nos. 5,594,121, 5,596,091; U.S. Patent Nos. 5,614,617; U.S. Patent Nos. 5,681,941; U.S. Patent Nos. 5,750,692; U.S. Patent Nos. 6,015,886; U.S. Patent Nos. 6,147,200; U.S. Patent Nos. 6,166,197; U.S. Patent Nos. 6,222,025; U.S. Patent Nos. 6,235,887; U.S. Patent Nos. 6,380,368; U.S. Patent Nos. 6,528,640; U.S. Patent Nos. 6,639,062; U.S. Patent Nos. 6,617,438; U.S. Patent Nos. 7,045,610; U.S. Patent Nos. 7,427,672; and U.S. Patent Nos. 7,495,088.

[0143] The RNA of the iRNA may also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a bridge between two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge linking two carbon atoms of a sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge links the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety that includes a further bridge linking the 2' and 4' carbons of the ribose moiety. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose in the 3' terminal structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides used in the polynucleotides of the present invention include, without limitation, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides containing a 4'-2' bridge.Examples of such 4'-2'-bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' (also known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogues; see, for example, U.S. Patent No. 8,278, Examples include: 4'-CH2-N(OCH3)-2' (and its analogues; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' [wherein R is H, C1-C12 alkyl, or a protecting group] (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and its analogues; see, e.g., U.S. Patent No. 8,278,426). All of the above-mentioned contents are incorporated herein by reference.

[0144] Further representative U.S. patents and U.S. patent application publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent No. 6,268,490; U.S. Patent No. 6,525,191; U.S. Patent No. 6,670,461; U.S. Patent No. 6,770,748; U.S. Patent No. 6,794,499; U.S. Patent No. 6,998,484; U.S. Patent No. 7,053,207; U.S. Patent No. 7,034,133; U.S. Patent No. 7,084,125; U.S. Patent No. 7,399,845. Examples include: U.S. Patent No. 7,427,672; U.S. Patent No. 7,569,686; U.S. Patent No. 7,741,457; U.S. Patent No. 8,022,193; U.S. Patent No. 8,030,467; U.S. Patent No. 8,278,425; U.S. Patent No. 8,278,426; U.S. Patent No. 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281 (each of which, in its entirety, is incorporated herein by reference).

[0145] Any of the aforementioned bicyclic nucleosides can be prepared to have one or more stereochemical sugar configurations, for example, α-L-ribofuranose and β-D-ribofuranose (see International Publication No. 99 / 14226).

[0146] The RNA of the iRNA may also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the restricted ethyl nucleotide is in an S configuration, which is referred to herein as “S-cEt”.

[0147] The iRNA of the present invention may also comprise one or more “conformational fixed nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. The CRN fixes the ribose ring to a stable configuration, increasing its hybridization affinity to mRNA. The linker is long enough to place oxygen in an optimal position in terms of stability and affinity, resulting in less puckering of the ribose ring.

[0148] Representative documents teaching some of the preparations of the CRNs described above include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and International Publication No. 2013 / 036868 (the entire contents of each of these are incorporated herein by reference).

[0149] One or more nucleotides of the iRNA of the present invention may also include hydroxymethyl-substituted nucleotides. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also known as an "unlocked nucleic acid" ("UNA") modification.

[0150] Representative U.S. publications that provide instruction on the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; U.S. Patent Application Publication No. 2013 / 0096289; U.S. Patent No. 2013 / 0011922; and U.S. Patent No. 2011 / 0313020 (the entire contents of each of these are incorporated herein by reference).

[0151] Potential stabilization modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyluridine-3''-phosphate, and the reversed base dT (idT). These modifications are disclosed in International Publication No. 2011 / 005861.

[0152] A. Modified iRNA containing the motif of the present invention In certain embodiments of the present invention, the double-stranded RNAi agent of the present invention includes an agent having chemical modifications as disclosed, for example, in International Publication No. 2013 / 075035, filed on November 16, 2012 (all of which are incorporated herein by reference). Excellent results can be obtained by introducing one or more identical modification motifs of three consecutive nucleotides to the sense strand and / or antisense strand of the RNAi agent, particularly to or near the cleavage site, as shown herein and in International Publication No. 2013 / 075035. In some embodiments, the sense strand and antisense strand of the RNAi agent may be fully modified in other cases. The introduction of these motifs disrupts the modification pattern of the sense strand and / or antisense strand, if present. The RNAi agent may optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0153] More specifically, surprisingly, it has been found that completely modifying the sense and antisense strands of a double-stranded RNAi agent to have one or more identical modification motifs of three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent significantly enhanced the gene silencing activity of the RNAi agent.

[0154] Accordingly, the present invention provides a double-stranded RNAi agent that has the ability to inhibit the expression of a target gene (i.e., the apolipoprotein C3 (APOC3) gene) in vivo. This RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 12 to 30 nucleotides long. For example, each strand may be 14 to 30 nucleotides long, 17 to 30 nucleotides long, 25 to 30 nucleotides long, 27 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 19 to 25 nucleotides long, 19 to 23 nucleotides long, 19 to 21 nucleotides long, 21 to 25 nucleotides long, or 21 to 23 nucleotides long.

[0155] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred to herein as the "RNAi agent." The double-stranded region of the RNAi agent may be 12–30 nucleotide pairs long. For example, the double-stranded region may 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 may be selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide lengths.

[0156] In one embodiment, the RNAi agent may contain one or more overhang regions and / or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhang may be 1 to 6 nucleotides long, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. The overhang may result from one strand being longer than the other, or from two strands of equal length being misaligned. The overhang may form a mismatch with the target mRNA, or it may be complementary to the target gene sequence, or it may be a different sequence. The first and second strands may also be joined together by additional bases, for example, to form a hairpin, or by other non-base linkers.

[0157] In one embodiment, the nucleotides in the overhang region of the RNAi agent may be, independently of any other, modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications such as 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 may be an overhang sequence at the end of either strand. The overhang may form a mismatch with the target mRNA, or it may be complementary to the target gene sequence, or it may be a different sequence.

[0158] The 5'- or 3'-overhangs of the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, one or more overhang regions include two nucleotides with a phosphorothioate in between, where the two nucleotides may be the same or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located on the antisense strand. In one embodiment, this 3'-overhang is located on the sense strand.

[0159] RNAi agents may contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, this single-stranded overhang may be located at the 3' end of the sense strand or at the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Although we do not wish to be constrained by theory, the asymmetric blunt end of the 5' end of the antisense strand and the 3' end overhang of the antisense strand are favorable for guide strands loaded into RISC processes.

[0160] In one embodiment, the RNAi agent is a 19-nucleotide-long double bluntmer, where the sense strand contains at least one of three 2'-F modification motifs on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one of three 2'-O-methyl modification motifs on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0161] In another embodiment, the RNAi agent is a 20-nucleotide-long double-ended bluntomer, where the sense strand contains at least one of three 2'-F modification motifs on three consecutive nucleotides from the 5' end at positions 8, 9, and 10. The antisense strand contains at least one 2'-O-methyl modification motif on three consecutive nucleotides from the 5' end at positions 11, 12, and 13.

[0162] In yet another embodiment, the RNAi agent is a 21-nucleotide-long double-ended bluntomer, where the sense strand contains at least one of three 2'-F modification motifs on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one of three 2'-O-methyl modification motifs on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0163] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on 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 on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the RNAi agent is blunt and the other end contains a 2-nucleotide overhang. Preferably, this 2-nucleotide overhang is at the 3' end of the antisense strand.

[0164] If a two-nucleotide overhang is located at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the three terminal nucleotides, where two of these nucleotides are the overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide bonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, all nucleotides on the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0165] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, where the sense strand is 25-30 nucleotides long and starts at the 5' terminal nucleotide (position 1), with positions 1-23 of the first strand containing at least 8 ribonucleotides; the antisense strand is 36-66 nucleotides long and starts at the 3' terminal nucleotide, forming a double helix with at least 8 ribonucleotides at positions opposite to positions 1-23 of the sense strand; where at least the 3' terminal nucleotide of the antisense strand does not pair with the sense strand, and up to 6 consecutive 3' terminal nucleotides do not pair with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; the 5' end of the antisense strand contains 10-30 consecutive nucleotides, which are opposite the sense strand. The sense strand does not pair, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; when the sense and antisense strands are aligned to achieve maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, reducing target gene expression when this double-stranded nucleic acid is introduced into mammalian cells; and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of these motifs is located at or near the cleavage site; the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0166] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, where the RNAi agent comprises a first strand having a length of 25 to 29 nucleotides, and a second strand having a length of 30 nucleotides or less and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; where the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the double-stranded region is at least 25 nucleotides long, and the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the length of the second strand, reducing target gene expression when introduced into mammalian cells, and dicer cleavage of the RNAi agent preferentially yields siRNA containing the 3' end of the second strand, thereby reducing target gene expression in mammals. Optionally, the RNAi agent further comprises a ligand.

[0167] In one embodiment, the sense strand of the RNAi agent contains at least one of three identical modification motifs on a triple nucleotide sequence, one of which is located at a cleavage site on the sense strand.

[0168] In one embodiment, the antisense strand of the RNAi agent may also contain at least one of three identical modification motifs on a triple nucleotide, where one of these motifs is located at or near the cleavage site of the antisense strand.

[0169] For RNAi agents with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically located at positions 10, 11, and 12 from the 5' end. Therefore, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 on the antisense strand (counting starts from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide from the 5' end within the double-stranded region of the antisense strand). The cleavage sites on the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0170] The sense strand of an RNAi agent may contain at least one motif of three identical modifications on a triple nucleotide at a cleavage site; and the antisense strand may have at least one motif of three identical modifications on a triple nucleotide at or near a cleavage site. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand may be aligned such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on 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 forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides, or all three nucleotides, may overlap.

[0171] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence. The first motif may be located at or near the cleavage site of the strand, and the other motifs may be wing modifications. The term “wing modification” as used herein refers to a motif located on a different part of the strand, separated from a motif located at or near the cleavage site of the same strand. A wing modification is either adjacent to the first motif or separated by at least one nucleotide. If the motifs are immediately adjacent to each other, their chemistry is different from that of the motifs; if the motifs are separated by one nucleotide or more, their chemistry may be the same or different. There may be two or more wing modifications. For example, if there are two wing modifications, each wing modification may be located on one side of the first motif at or near the cleavage site, or on both sides of the read motif.

[0172] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence, at least one of which may be located at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications aligned in a similar manner to the wing modifications that may be present on the sense strand.

[0173] In one embodiment, the wing modification on the sense or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0174] In another embodiment, the wing modification on the sense or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides in the double-stranded region located at the 3' end, 5' end, or both ends of the strand.

[0175] If the sense strand and antisense strand of an RNAi agent each contain at least one wing modification, these wing modifications may be located at the same ends of the double-stranded region and have an overlap of 1, 2, or 3 nucleotides.

[0176] If the sense strand and antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and antisense strand may be aligned such that two modifications from one strand are located at one end of the double-stranded region, with a 1, 2, or 3 nucleotide overlap; two modifications from one strand are located at the other end of the double-stranded region, with a 1, 2, or 3 nucleotide overlap; or the two modifications on one strand are located on both sides of the read motif, with a 1, 2, or 3 nucleotide overlap in the double-stranded region.

[0177] In one embodiment, any nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, may be modified. Each nucleotide may be modified by the same or different modifications, which may include: one or more modifications of one or both of the unbound phosphate oxygen and / or bound phosphate oxygen; modifications of components of the ribose sugar, e.g., the 2' hydroxyl on the ribose sugar; large-scale substitution of the phosphate moiety by a "dephospho" linker; modifications or substitutions of naturally occurring bases; and substitutions or modifications of the ribose phosphate backbone.

[0178] Since nucleic acids are polymers of subunits, many modifications are located at repeating positions within the nucleic acid, such as modifications of bases, or phosphate moieties, or unbound oxygen atoms of phosphate moieties. In some cases, modifications may be present at all target positions within the nucleic acid, but this is not often the case. For example, modifications may be present only at the 3' or 5' end, or only in the terminal region, e.g., at a position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may be present in double-stranded regions, single-stranded regions, or both. Modifications may be present only in the double-stranded regions of RNA, or only in the single-stranded regions of RNA. For example, a phosphorothioate modification at an unbound oxygen position may be present only at one or both ends, or only in the terminal region, e.g., at a position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 nucleotides of the strand, or in both double-stranded and single-stranded regions, especially at the ends. One or more 5' ends may be phosphorylated.

[0179] For example, to enhance stability, the overhang may contain specific bases, or it may contain modified nucleotides or nucleotide surrogates in single-stranded overhangs, e.g., 5' or 3' overhangs, or both. For example, it may be desirable to contain purine nucleotides in the overhang. In some embodiments, all or some of these bases in the 3' or 5' overhang may be modified by modifications, for example, those described herein. Modifications may include modifications known in the art, e.g., deoxyribonucleotides modified in place of ribosaccharides in nucleic acid bases, modifications at the 2' position of ribose sugars by the use of 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl, and modifications at phosphate groups, e.g., the use of phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0180] In one embodiment, each residue in the sense and antisense chains is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0181] Typically, at least two different modifications are present on the sense and antisense chains. These two modifications may include 2'-O-methyl or 2'-fluoro modifications.

[0182] In one embodiment, Na and / or Nb include alternating pattern modifications. The term “alternating motif,” as used herein, refers to a motif having one or more modifications, where each modification lies on alternating nucleotides on one of the strands. Alternating nucleotides may refer to nucleotides every other nucleotide, every three nucleotides, or similar patterns. 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.

[0183] The types of modifications included in the alternating motif may be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand may be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0184] In one embodiment, the RNAi agent of the present invention includes a shift in the modification pattern of the alternating motif on the sense strand relative to the modification pattern of the alternating motif on the antisense strand. This shift may be such that modified groups of nucleotides on the sense strand correspond to differently modified groups of nucleotides on the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double helix, the alternating motif on the sense strand within the double helix region may begin with "ABABAB" at 5'-3' of the strand, and the alternating motif on the antisense strand may begin with "BABABA" at 5'-3' of the strand. As another example, the alternating motif on the sense strand within the double helix region may begin with "AABBAABB" at 5'-3' of the strand, and the alternating motif on the antisense strand may begin with "BBAABBAA" at 5'-3' of the strand, thus there is a complete or partial shift in the modification patterns between the sense strand and the antisense strand.

[0185] In one embodiment, the RNAi agent includes a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the sense strand, which initially has a shift relative to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the antisense strand, i.e., a 2'-O-methyl modified nucleotide on the sense strand is base-paired with a 2'-F modified nucleotide on the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

[0186] Introducing one or more identical modification motifs of three consecutive nucleotides into the sense strand and / or antisense strand disrupts the original modification pattern present in the sense strand and / or antisense strand. This disruption of the modification pattern of the sense strand and / or antisense strand, caused by introducing one or more identical modification motifs of three consecutive nucleotides into the sense strand and / or antisense strand, unexpectedly enhances gene silencing activity against the target gene.

[0187] In one embodiment, if a motif of three identical modifications on a triple nucleotide is introduced to any of these chains, the modifications of the nucleotides adjacent to the motif are different from the modifications of the motif. For example, the portion of the sequence containing the motif is "...NaYYYNb..." [wherein "Y" represents the modifications of the motif of three identical modifications on a triple nucleotide, and "Na" and "Nb" represent modifications to the nucleotides adjacent to the motif "YYY" that are different from the modifications of Y, and Na and Nb may be the same or different modifications]. Alternatively, Na and / or Nb may or may not be present when a wing modification is present.

[0188] The RNAi agent may further contain at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification may be present on any nucleotide at any position on the sense strand, the antisense strand, or both strands. For example, the internucleotide bond modification may be present on any nucleotide on the sense strand and / or antisense strand; each internucleotide bond modification may be present in an alternating pattern on the sense strand and / or antisense strand; or both the sense strand and the antisense strand may contain internucleotide bond modifications in an alternating pattern. The alternating pattern of internucleotide bond modifications on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide bond modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide bond modifications on the antisense strand. In one embodiment, a double-standed RNAi agent contains 6 to 8 phosphorothioate internucleotide bonds. In one embodiment, the antisense strand includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end, and the sense strand includes at least two phosphorothioate nucleotide interlinks at either its 5' or 3' end.

[0189] In one embodiment, the RNAi includes phosphorothioate or methylphosphonate internucleotide linkage modifications in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between them. The internucleotide linkage modifications may also be made to link the overhang nucleotides to terminal paired nucleotides in the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked via phosphorothioate or methylphosphonate internucleotide linkages, and optionally there may be further phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotides to the paired nucleotides adjacent to the overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhang nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhang nucleotides. These three terminal nucleotides may be located 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.

[0190] In one embodiment, the two-nucleotide overhang is located at the 3' end of the antisense strand, with two phosphorothioate internucleotide bonds between the three terminal nucleotides, where two of these nucleotides are the overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotides. Optionally, the RNAi agent may further have two phosphorothioate internucleotide bonds between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0191] In one embodiment, the RNAi agent contains mismatches with targets within the double helix, or combinations thereof. Mismatches may be present in overhang regions or double helix regions. Base pairs may be ranked based on their tendency to promote dissociation or dissolution (for example, regarding the free energy of association or dissociation of particular pairings, the simplest method is to examine each individual pair base, however, the following adjacent 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; and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (as described in other parts of this specification) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings containing universal bases are preferred over canonical pairings.

[0192] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs from the 5' end within the double-strand region of the antisense strand, independently selected from the group of A:U, G:U, I:C, and mismatch pairs, such as non-canonical or non-canonical pairings or pairings containing universal bases, thereby promoting the dissociation of the antisense strand at the 5' end of the double helix.

[0193] In one embodiment, the nucleotide at position 1 from the 5' end within the double-stranded region 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 from the 5' end within the double-stranded region of the antisense strand is an AU base pair. For example, the first base pair from the 5' end within the double-stranded region of the antisense strand is an AU base pair.

[0194] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, the 3' ends of the sense strand and / or antisense strand have a short sequence of deoxythymine nucleotides, for example, two dT nucleotides.

[0195] In one embodiment, the sense strand sequence is given by formula (I): 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'(I) [In formula: i and j are independently either 0 or 1; p and q are each independently between 0 and 6; Each Na independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides; Each Nb independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Each np and nq independently represents an overhang nucleotide; Here, Nb and Y do not have the same modifications; and XXX, YYY, and ZZZ can each be represented independently by one motif of three identical modifications on a triple nucleotide sequence. Preferably, all YYY are 2'-F modified nucleotides.

[0196] In one embodiment, Na and / or Nb include alternating pattern modifications.

[0197] In one embodiment, the YYY motif is located at or near the sense strand cleavage site. For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may be located at or near the sense strand cleavage site (e.g., it may be located at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11,12 or 11,12,13) ​​(counting begins from the first nucleotide from the 5' end; or optionally, counting begins from the first paired nucleotide from the 5' end within the double-stranded region).

[0198] 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. Therefore, the sense chain is given by the following equation: 5'np-Na-YYY-Nb-ZZZ-Na-nq3'(Ib); 5'np-Na-XXX-Nb-YYY-Na-nq3'(Ic); or 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3'(Id) It can be represented by [this].

[0199] When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0200] When the sense strand is represented by formula (Ic), Nb represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0201] When the sense strand is represented by formula (Id), each Nb independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6. Each Na independently may represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0202] Each of X, Y, and Z may be the same as or different from one another.

[0203] In other embodiments, i is 0 and j is 0, and the sense chain is given by the formula: 5'np-Na-YYY-Na-nq3'(Ia) It can be represented by:

[0204] When the sense strand is represented by formula (Ia), each Na independently may represent an oligonucleotide sequence containing 2–20, 2–15, or 2–10 modified nucleotides.

[0205] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5'nq'-Na'-(Z'Z'Z')k-Nb'-Y'Y'Y'-Nb'-(X'X'X')l-Na'-np'3'(II) [In formula: k and l are independently either 0 or 1; p' and q' are each independently between 0 and 6; Each Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides; Each Nb' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Each np' and nq' independently represents an overhang nucleotide; Here, Nb' and Y' do not have the same modification; and X'X'X', Y'Y'Y', and Z'Z'Z' can each be represented independently by a single motif of three identical modifications on a triple nucleotide sequence.

[0206] In one embodiment, Na' and / or Nb' include alternating pattern modifications.

[0207] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, when an RNAi agent has a double-stranded region of 17-23 nucleotides in length, the Y'Y'Y' motif may be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand (counting starts from the first nucleotide from the 5' end; or optionally, counting starts from the first paired nucleotide from the 5' end within the double-stranded region). Preferably, the Y'Y'Y' motif is located at positions 11, 12, 13.

[0208] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0209] 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.

[0210] Therefore, the antisense chain is given by the following formula: 5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Na'-np'3'(IIb); 5'nq'-Na'-Y'Y'Y'-Nb'-X'X'X'-np'3'(IIc); or 5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Nb'-X'X'X'-Na'-np'3'(IId) It can be represented by:

[0211] When the antisense strand is represented by formula (IIb), Nb' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0212] When the antisense strand is represented by formula (IIc), Nb' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0213] When the antisense strand is represented by formula (IId), each Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6.

[0214] In another embodiment, k is 0 and l is 0, and the antisense chain is given by the formula: 5'np'-Na'-Y'Y'Y'-Na'-nq'3'(Ia) It can be represented by:

[0215] When the antisense strand is represented by formula (IIa), each Na' independently represents an oligonucleotide sequence containing 2–20, 2–15, or 2–10 modified nucleotides.

[0216] Each of X', Y', and Z' may be the same as or different from the others.

[0217] Each nucleotide in the sense and antisense strands may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands may be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in detail, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0218] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif located at positions 9, 10, and 11 of the strand when the double-stranded region is 21nt (counting starts from the first nucleotide from the 5' end, or optionally, the counting starts from the first paired nucleotide from the 5' end within the double-stranded region); and Y represents a 2'-F modification. The sense strand may further contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0219] In one embodiment, the antisense strand may contain a Y'Y'Y' motif located at positions 11, 12, and 13 of the strand (counting starting from the first nucleotide from the 5' end, or optionally, starting from the first paired nucleotide from the 5' end within the double-stranded region); and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0220] A sense chain represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense chain represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

[0221] Therefore, the RNAi agent used in the method of the present invention may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the RNAi double helix is ​​given by formula (III): Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3' Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5' (III) [In formula: i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; Each Na and Na' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides; Each Nb and Nb' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Here, each np', np, nq', and nq (each of which may or may not be present) independently represents an overhang nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It is represented by [this].

[0222] 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.

[0223] The following is an example of a sense strand and antisense strand combination that forms an RNAi double helix: 5'np-Na-YYY-Na-nq3' 3'np'-Na'-Y'Y'Y'-Na'nq'5' (IIIa) 5'np-Na-YYY-Nb-ZZZ-Na-nq3' 3'np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5' (IIIb) 5'np-Na-XXX-Nb-YYY-Na-nq3' 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq'5' (IIIc) 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3' 3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq'5' (IIId)

[0224] When an RNAi agent is represented by formula (IIIa), each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0225] When an RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0226] When an RNAi agent is represented by formula (IIIc), each Nb and Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0227] When an RNAi agent is represented by formula (IIId), each Nb and Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na and Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na', Nb, and Nb' independently contains alternating modification patterns.

[0228] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), X, Y, and Z may be the same as or different from each other.

[0229] When an RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y' nucleotides; or at least two of the Y nucleotides may form a base pair with the corresponding Y' nucleotide; or all three of the Y nucleotides may form a base pair with the corresponding Y' nucleotide.

[0230] When an RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z' nucleotides; or at least two of the Z nucleotides may form a base pair with the corresponding Z' nucleotide; or all three of the Z nucleotides may form a base pair with the corresponding Z' nucleotide.

[0231] When an RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X' nucleotides; or at least two of the X nucleotides may form a base pair with the corresponding X' nucleotide; or all three of the X nucleotides may form a base pair with the corresponding X' nucleotide.

[0232] In one embodiment, modifications on the Y nucleotide differ from modifications on the Y' nucleotide, modifications on the Z nucleotide differ from modifications on the Z' nucleotide, and / or modifications on the X nucleotide differ from modifications on the X' nucleotide.

[0233] In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, with np'>0 and at least one np' linked to an adjacent nucleotide via a phosphorothioate bond. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, with np'>0 and at least one np' linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives (described below) added via a divalent or trivalent branched linker. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives added via a divalent or trivalent branched linker.

[0234] In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives added via a divalent or trivalent branched linker.

[0235] In one embodiment, the RNAi agent is a multimer containing at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), which are linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further contains ligands. Each double helix may target the same gene or two different genes; or each double helix may target the same gene at two different target sites.

[0236] In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double helixes are linked by linkers. The linkers may be cleavable or non-cleavable. Optionally, the multimer further contains ligands. Each double helix may target the same gene or two different genes; or each double helix may target the same gene at two different target sites.

[0237] In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are ligated together at one or both of their 5' and 3' ends and optionally coupled to a ligand. Each of these agents may target the same gene or two different genes; or each of these agents may target the same gene at two different target sites.

[0238] In certain embodiments, the RNAi agents of the invention may contain a small number of nucleotides containing 2'-fluoro modifications, for example, 10 or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides containing 2'-fluoro modifications. In a specific embodiment, the RNAi agent of the invention contains 10 nucleotides containing 2'-fluoro modifications, for example, 4 nucleotides containing 2'-fluoro modifications in the sense strand and 6 nucleotides containing 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the invention contains 6 nucleotides containing 2'-fluoro modifications, for example, 4 nucleotides containing 2'-fluoro modifications in the sense strand and 2 nucleotides containing 2'-fluoro modifications in the antisense strand.

[0239] In other embodiments, the RNAi agents of the invention may contain a very small number of nucleotides containing 2'-fluoro modifications, for example, 2 or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 2, 1, or 0 nucleotides containing 2'-fluoro modifications. In a specific embodiment, the RNAi agent may contain 2 nucleotides containing 2'-fluoro modifications, for example, 0 nucleotides containing 2'-fluoro modifications in the sense strand and 2 nucleotides containing 2'-fluoro modifications in the antisense strand.

[0240] A variety of documents describe multimer RNAi agents that can be used in the methods of the invention. Such documents include WO 2007 / 091269 pamphlet, US Patent No. 7858769 specification, WO 2010 / 141511 pamphlet, WO 2007 / 117686 pamphlet, WO 2009 / 014887 pamphlet, and WO 2011 / 031520 pamphlet (the entire contents of each of which are hereby incorporated by reference herein).

[0241] As described in more detail below, an RNAi agent containing the conjugation of one or more carbohydrate moieties with the RNAi agent can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety can be added 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, for example, a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is added. A ribonucleotide subunit in which the ribose sugar of the subunit is thus replaced is referred to herein as a ribose-substituted modified subunit (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or it may be a heterocyclic system, i.e., one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, and sulfur. The cyclic carrier may be a monocyclic system, or it may contain two or more rings, for example, a fused ring. The cyclic carrier may be a fully saturated cyclic system, or it may contain one or more double bonds.

[0242] Ligands can be attached to polynucleotides via a carrier. The carrier comprises (i) at least one “skeletal linkage site,” preferably two “skeletal linkage sites,” and (ii) at least one “tethering linkage site.” “Skeletal linkage site,” as used herein, refers to a functional group, e.g., a hydroxyl group, or generally, a linkage available and suitable for incorporating the carrier into a skeleton, e.g., the phosphate skeleton of ribonucleic acid, or a modified phosphate skeleton, e.g., a sulfur-containing skeleton. “Tethering linkage site” (TAP) refers, in some embodiments, to a constituent ring atom of a cyclic carrier that ties a portion of choice, e.g., a carbon atom or heteroatom (different from the atom providing the skeletal linkage site). This portion may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Optionally, the portion of choice is attached to the cyclic carrier by intervening tethering. Thus, the cyclic carrier often contains a functional group, e.g., an amino group, or generally provides a linkage suitable for incorporating or tethering another chemical substance, e.g., a ligand, into the constituent ring.

[0243] The RNAi agent may be conjugated with a ligand via a carrier, where the carrier can be a cyclic group or an acyclic 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 acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0244] In certain specific embodiments, the RNAi agent used in the method of the present invention is an agent selected from the group of agents listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. These agents may further contain a ligand.

[0245] IV. Ligand-conjugated iRNA Another modification of the iRNA of the present invention involves chemically linking one or more ligands, moieties, or complexes to the RNA, which enhance the activity, cell distribution, or intracellular uptake of the iRNA. Such parts include lipid portions such as the cholesterol portion (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770) and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); and thioethers such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO Aliphatic chains such as J,1991,10:1111-1118;Kabanov et al.,FEBS Lett.,1990,259:327-330;Svinarchuk et al.,Biochimie,1993,75:49-54); phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654;Shea et al.,Nucl.Acids Res.,1990,18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969-973; or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys.Examples include, but are not limited to, octadecylamine or the hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0246] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides improved affinity to selected targets, such as molecules, cells or cell types, compartments such as intracellular or organelle compartments, tissues or organs or regions of the body, compared to chemical species in which such ligand is absent. The preferred ligand does not participate in double-strand pairing in the double-stranded nucleic acid.

[0247] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or α-helical peptides.

[0248] The ligand may also include targeting groups such as antibodies that bind to specific cell types, such as kidney cells, or cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins. The targeting groups may be thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetylglucosamine (gulucoseamine), polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimetic compounds.

[0249] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithoglycerol Examples include lic acid, O3-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine) and peptide complexes (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole complexes, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, or AP.

[0250] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for a co-ligand; or antibodies, such as antibodies that bind to a specified cell type, such as liver cells. Ligands may also include hormones and hormone receptors. They may also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide chemical species such as polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, or polyhydric fucose. Ligands may be lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators, for example.

[0251] Ligands can be substances such as drugs that can increase the uptake of iRNA agents into cells by disrupting, for example, the cellular microtubules, microfibrils, and / or intermediate filaments, or by disrupting the cellular cytoskeleton. Drugs may include, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0252] In some embodiments, the ligands attached to iRNAs described herein refer to pharmacokinetic modifiers (PK modifiers). Examples of PK modifiers include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, and vitamins. Exemplary PK modifiers include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate bonds are also known to bind to serum proteins, and therefore, for example, short-chain oligonucleotides such as approximately 5-base, 10-base, 15-base, or 20-base oligonucleotides containing multiple phosphorothioate bonds in the main chain are also suitable as ligands (e.g., as PK modulating ligands) in the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0253] The ligand-conjugated oligonucleotides of the present invention may be synthesized using oligonucleotides having pendant-reactive functional groups, such as those derived from the addition of a binding molecule onto the oligonucleotide (described below). These reactive oligonucleotides may be reacted directly with commercially available ligands, synthesized ligands having any of the various protecting groups, or ligands having a binding site to which attachment is possible.

[0254] The oligonucleotides used in the complexes of the present invention may, conveniently and conventionally, be produced through well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including Applied Biosystems (Foster City, Calif.). Alternatively, any other means known in the art for such synthesis may be used. It is also known that other oligonucleotides, such as phosphorothioates and alkylated derivatives, can be prepared using similar techniques.

[0255] In the ligand-conjugated oligonucleotides and sequence-specific binding nucleosides containing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotides or nucleoside precursors, nucleotides or nucleoside complex precursors already containing binding sites, ligand-nucleotide or nucleoside complex precursors already containing ligand molecules, or basic units containing non-nucleoside ligands.

[0256] When using a nucleotide complex precursor that already has a binding site, the synthesis of a sequence-specific bound nucleoside is typically completed, and then the ligand molecule reacts with the binding site to produce a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or bound nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside complexes, in addition to standard and non-standard phosphoramidites that are commercially available and conventionally used in oligonucleotide synthesis.

[0257] A. Lipid complexes In one embodiment, the ligand or complex is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the complex to target tissues, such as non-renal target tissues of the body. Target tissues, for example, could be the liver, including hepatic parenchymal cells. Other molecules capable of binding to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the degradation resistance of the complex, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate the binding of serum proteins, such as HSA.

[0258] Lipid-based ligands can be used for inhibition, for example, by controlling the binding of the complex to target tissues. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the complex to the kidneys.

[0259] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the complex is distributed to non-renal tissues. However, the affinity is preferably not so strong as to prevent the HSA ligand binding from being reversed.

[0260] In another preferred embodiment, the lipid-based ligand binds weakly to or does not bind at all to the HSA so that the complex is preferably distributed to the kidney. Other portions that target renal cells may also be used instead of, or in addition to, the lipid-based ligand.

[0261] In another aspect, the ligand is a moiety such as a vitamin that is taken up by target cells such as proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant forms such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

[0262] B. Cell-Penetrating Agents In another aspect, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the cell-penetrating agent is amphiphilic. Exemplary cell-penetrating agents are peptides such as tat or antennapedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidomimetics, reverse isomers, non-peptides or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having hydrophilic and hydrophobic phases.

[0263] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can fold into a defined three-dimensional structure similar to a natural peptide. The addition of peptides and peptidomimetics to the iRNA agent can affect the pharmacokinetic distribution of the iRNA, such as by promoting cell recognition and uptake. The peptide or peptidomimetic moiety can be, for example, about 5 to 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0264] Peptides or peptide mimetic drugs may be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, a bound peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane-transfer sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 24). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 25)) may also be target moieties. The peptide moiety may be a “delivery” peptide capable of transporting a number of polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 26)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 27)) have been shown to function as delivery peptides. Peptides or peptide mimetic drugs 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., Nature, 354:82-84, 1991). For cell targeting purposes, examples of peptides or peptide mimetic drugs anchored to dsRNA activators via incorporated monomer units include arginine-glycine-aspartate (RGD) peptides or RGD mimetic drugs. The peptide moiety can range in length from approximately 5 to 40 amino acids. The peptide moiety may have structural modifications that increase stability or induce conformational properties. Any of the structural modifications described below may be used.

[0265] The RGD peptides used in the compositions and methods of the present invention may be linear or cyclic, and may be modified, for example, by glycosylation or methylation to facilitate targeting to specific tissues. Examples of RGD-containing peptides and peptide mimetic agents include D-amino acids and synthetic RGD mimetic agents. In addition to RGD, other moieties that target integrin ligands may be used. Preferred ligand complexes target PECAM-1 or VEGF.

[0266] "Cell-permeable peptides" can penetrate cells such as microbial cells, including bacterial or fungal cells, or mammalian cells, including human cells. Microbial cell-permeable peptides may be, for example, α-helical linear peptides (e.g., LL-37 or ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphimotic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0267] C. Carbohydrate complex In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, “carbohydrate” means a carbohydrate itself, which consists of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound which has as part a carbohydrate portion consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) each having an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Examples of specific monosaccharides include sugars with a monosaccharide count of AGT or higher (e.g., AGT, C6, C7, or C8); disaccharides include sugars having two or three monosaccharide units (e.g., AGT, C6, C7, or C8).

[0268] In one embodiment, the carbohydrate conjugate used in the composition and method of the present invention is a monosaccharide. In one embodiment, the monosaccharide is [ka] These include N-acetylgalactosamine.

[0269] In another embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0270] Other representative carbohydrate conjugates used in the embodiments described herein include, but are not limited to, [ka] (Formula XXIII) [If one of X or Y is an oligonucleotide, the other is hydrogen] is included.

[0271] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, including, but not limited to, PK modulators and / or cell-permeable peptides.

[0272] Further carbohydrate conjugates suitable for use in the present invention are those described in International Publication No. 2014 / 179620 and International Publication No. 2014 / 179627 (all of which are incorporated herein by reference).

[0273] D. Linker In some embodiments, the complexes or ligands described herein may be attached to the iRNA oligonucleotide by various linkers, which may be cleavable or incleavable.

[0274] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, such as by covalently bonding two parts of the compound together.Linkers are typically directly bonded, or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or substituted or unsubstituted alkyls, substituted or unsubstituted alkenyls, substituted or unsubstituted alkynyls, arylalkyls, arylalkenyls, arylalkynyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryls, heteroaryl, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls, alkenylheteroarylalkyls, alkenylheteroarylalkenyls, alkenylheteroaryl Alkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heterocyclylalkenyl, alkyl heterocyclylalkynyl (alkyl hererocyclylalkynyl), alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl The atomic chain includes, but is not limited to, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl, and one or more methylene groups therein may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle (wherein R8 is hydrogen, acyl, aliphatic, or substituted aliphatic).In one embodiment, the linker consists of approximately 1 to 24 atoms, 2 to 24 atoms, 3 to 24 atoms, 4 to 24 atoms, 5 to 24 atoms, 6 to 24 atoms, 6 to 18 atoms, 7 to 18 atoms, 7 to 17 atoms, 8 to 17 atoms, 6 to 16 atoms, 7 to 16 atoms, or 8 to 16 atoms.

[0275] The cleavable linking group is sufficiently stable outside the cell but is cleaved upon entry into the target cell, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more, or at least about 100 times faster in the target cell or under a first standard condition (which may be selected to mimic or be equivalent to intracellular conditions) than in the target blood or under a second standard condition (which may be selected to mimic or be equivalent to conditions found in blood or serum).

[0276] Cleavable linkers are susceptible to the influence of cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common in cells than in serum or blood, or are found at higher levels or activity. Examples of such degradable agents include oxidative or reductases or reducing agents such as mercaptans present in cells that can degrade redox-cleavable linkers by reduction, and redox-selected or non-substrate-specific redox agents selected for specific substrates; esterases; agents that can create acidic environments, such as endosomes or those that result in a pH of 5 or less; and enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0277] Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. While human serum has a pH of 7.4, the mean intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a favorable pH, thereby releasing cationic lipids from ligands within the cell or to desired compartments of the cell.

[0278] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into a linker may depend on the target cell. For example, a ligand targeting the liver may link to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and therefore linkers are cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0279] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synovial cells.

[0280] Generally, the suitability of candidate cleavable linkers can be evaluated by testing the ability of a degrading agent (condition) to cleave the candidate linker. It is also desirable to test the candidate cleavable linker's resistance to cleavage in the blood or in contact with other non-target tissues. Therefore, the relative susceptibility to cleavage between a first and second condition can be determined, with the first condition selected to demonstrate cleavage in target cells and the second condition selected to demonstrate cleavage in other tissues or in biological fluids such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or in whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and then confirm it with further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0281] i. Redox-cleavable linking groups In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). Methods described herein can be relied upon to determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent. For example, a candidate may be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. One candidate compound is cleaved by up to about 10% in blood. In other embodiments, useful candidate compounds are degraded at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using a standard enzyme kinetics assay under conditions selected to mimic an extracellular medium, compared to conditions selected to mimic an extracellular medium.

[0282] ii. Phosphate-based cleavable linking groups In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group can be cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves the phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0283] iii. Acid-cleavable linking group In another embodiment, the cleavable linker includes an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, or less) or by an active agent such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles such as endosomes and lysosomes can provide an environment for cleaving acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable linking group may have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when the carbon is attached to the oxygen of the ester (alkoxy group), it is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0284] iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. The ester-based cleavable linking group is cleaved in the cell by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkylylene groups. The ester-based cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0285] v. Peptide-based cleavage groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved in cells by enzymes such as peptidases and proteases. The peptide-based cleavable linking group is a peptide bond, which is formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable linking group does not contain an amide group (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond that is formed between amino acids to produce peptides and proteins. The peptide-based cleavable linking group is generally limited to peptide bonds (i.e., amide bonds) that are formed between amino acids to produce peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linking group has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0286] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates containing the linker of the composition and method of the present invention include, but are not limited to, [ka] [ka] [ka] [If one of X or Y is an oligonucleotide, the other is hydrogen.]

[0287] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker.

[0288] In one embodiment, the dsRNA of the present invention is given by formulas (XXXII) to (XXXV): [ka] [In formula: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent a number from 0 to 20, and their repeating units may be the same or different; P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently present in either absence or CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, and Q5C are, independently of each other, non-existent, alkylenes, or substituted alkylenes, where one or more methylene groups may be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R')=C(R"), C≡C, or C(O); R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, and R5C each exist independently, either not existing or not existing, as NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=NO. [ka] or heterocycline; L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B, and L5C represent ligands; that is, each independently represents a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide in each presence; and Ra is H or an amino acid side chain. These are conjugated to a divalent or trivalent branched linker selected from the group of structures shown in formula (XXXV). Trivalent conjugated GalNAc derivatives are given by formula (XXXV) [ka] It is particularly useful for use in RNAi agents that inhibit the expression of target genes, such as those in the formula [wherein L5A, L5B, and L5C represent monosaccharides such as GalNAc derivatives].

[0289] Suitable divalent and trivalent branched linker groups conjugated to GalNAc derivatives include, but are not limited to, the structures listed above as formulas II, VII, XI, X, and XIII.

[0290] Representative U.S. patents teaching the preparation of RNA complexes are, as are incorporated herein by reference in their entirety: U.S. Patent No. 4,828,979; U.S. Patent No. 4,948,882; U.S. Patent No. 5,218,105; U.S. Patent No. 5,525,465; U.S. Patent No. 5,541,313; U.S. Patent No. 5,545,730; U.S. Patent No. 5,552,538; U.S. Patent No. 5,578,717; U.S. Patent No. 5,580,731; and U.S. Patent No. 5,591,584. Detailed Statement; U.S. Patent No. 5,109,124; U.S. Patent No. 5,118,802; U.S. Patent No. 5,138,045; U.S. Patent No. 5,414,077; U.S. Patent No. 5,486,603; U.S. Patent No. 5,512,439; U.S. Patent No. 5,578,718; U.S. Patent No. 5,608,046; U.S. Patent No. 4,587,044; U.S. Patent No. 4,605,735; U.S. Patent No. 4,667,025; U.S. Patent No. 4,762,779; U.S. Patent No. 4,789,737 Details Document; U.S. Patent No. 4,824,941; U.S. Patent No. 4,835,263; U.S. Patent No. 4,876,335; U.S. Patent No. 4,904,582; U.S. Patent No. 4,958,013; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,245,022; U.S. Patent No. 5,254,469 U.S. Patent No. 5,258,506; U.S. Patent No. 5,262,536; U.S. Patent No. 5,272,250; U.S. Patent No. 5,292,873; U.S. Patent No. 5,317,098; U.S. Patent No. 5,371,241, U.S. Patent No. 5,391,723; U.S. Patent No. 5,416,203, U.S. Patent No. 5,451,463; U.S. Patent No. 5,510,475; U.S. Patent No. 5,512,667; U.S. Patent No. 5,514,785; U.S. Patent No. 5,565,552;U.S. Patent Nos. 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646 and 8,106,022 are examples, but are not limited to these.

[0291] It is not necessary for all positions in a given compound to be uniformly modified; in fact, two or more of the aforementioned modifications can be incorporated into a single compound, or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0292] In the context of this invention, a "chimeric" iRNA compound or "chimeras" is an iRNA compound, preferably a dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to give the iRNA increased resistance to nuclease degradation, increased intracellular uptake, and / or increased binding affinity to a target nucleic acid. The additional region of the iRNA may act as an enzyme substrate capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded molecule. Therefore, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region, comparable results are often obtained with shorter iRNAs. Cleavage of RNA targets can conventionally be detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0293] In some cases, the RNA of iRNA can be modified with non-ligand groups. Several non-ligand molecules are conjugated to iRNA to enhance its activity, cell distribution, or intracellular uptake, and procedures for performing such conjugations are available in the academic literature.These non-ligand portions include lipid portions such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids Aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & It contains Nucleotides (1995, 14:969), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents teaching the preparation of such RNA complexes are listed above. A typical conjugation protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group is then reacted with a molecule that conjugates the amino group using an appropriate coupling or activating reagent. The conjugation reaction can be carried out in the solution phase while the RNA is still bound to a solid support, or following RNA cleavage. Purification of the RNA complex by HPLC typically yields a pure complex.

[0294] V. Delivery of iRNA by invention For example, delivery of the iRNA of the present invention to cells, such as cells within a subject (e.g., a subject with APOC3-related disease or other subject requiring it), can be achieved in several different ways. For example, delivery may be carried out by contacting cells with the iRNA of the present invention, either in vitro or in vivo. In vivo delivery may also be carried out directly by administering a composition containing the iRNA, such as dsRNA, to the subject. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode and induce the expression of the iRNA. These alternatives are discussed further below.

[0295] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNA of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, whose entire contents are incorporated herein by reference). For in vivo delivery, factors to be considered for delivering the iRNA molecule include, for example, the biological stability of the delivery molecule, prevention of nonspecific effects, and accumulation of the delivery molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or transplantation into tissue or local administration of the formulation. Local administration to the treatment site maximizes the local concentration of the active substance, limits exposure of systemic tissues to the active substance which could otherwise be harmed or degraded by the active substance, and allows for administration of lower total doses of the iRNA molecule. Several studies have shown successful gene product knockdown when iRNA is administered locally. For example, intravitreal injection of VEGF dsRNA 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) both demonstrated the prevention of neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol.Ther.11:267-274) and extended the survival time of mice with tumors (Kim, WJ., et al (2006) Mol.Ther.14:343-350; Li, S., et al (2007) Mol.Ther.15:515-523).RNA interference can be administered to the CNS 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 to the lungs by intranasal administration (Howard, KA., et al. (2006) Mol.Ther.14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med.11:50-55) Successful local delivery has been demonstrated. To treat diseases, or to administer iRNA systemically, RNA can be modified or, alternatively, delivered using drug delivery systems; both methods act to prevent rapid degradation of dsRNA by endogenous and exonucleases. Modification of RNA or pharmaceutical carriers can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical bonding of lipophilic groups such as cholesterol to enhance intracellular uptake and prevent degradation. For example, when an iRNA that counteracts ApoB, which is conjugated to the lipophilic cholesterol portion, was systemically injected into mice, apoB mRNA knockdown was induced in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). The conjugation of iRNA to aptamers has been shown to suppress tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNA may be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of the iRNA molecule (which is negatively charged) and also enhance interactions with the negatively charged cell membrane, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may be induced to bind to iRNA or form vesicles or micelles that enclose the iRNA (see, e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of iRNA when administered systemically. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), cited above; Verma, UN., et al (2003), cited above), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441:111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME., et al. Examples include al (2008) Pharm. Res., published online on August 16; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, for systemic administration, the iRNA forms a complex with cyclodextrin. Methods of administering iRNA and cyclodextrin and pharmaceutical compositions are described in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0296] A. Vector encoding the iRNA of the present invention APOC3 gene-targeting iRNAs 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 Publication No. 00 / 22113; Conrad, International Publication No. 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a few hours to several weeks) or persistent (from several weeks to several months or more), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which may be embedded or non-embedded vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (see, for example, Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0297] Individual iRNA strands or strand groups can be transcribed from a promoter on an expression vector. When expressing two separate strands to generate, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (e.g., by transfusion or infection). Alternatively, the individual strands of the dsRNA can be transcribed by promoters located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence, such that the dsRNA has a stem-loop structure.

[0298] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for iRNA expression described herein can be generated using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial suppliers. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors may include systemic administration, such as intravenous or intramuscular administration; administration to target cells explanted from a patient and subsequent reintroduction into the patient; or any other means that allows introduction into desired target cells.

[0299] iRNA expression plasmids can be translocated into target cells as complexes with cationic lipid carriers (e.g., oligofectamines) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid translocation for iRNA-mediated knockdown, targeting different regions of the target RNA over a period of more than one week, is also explored in this invention. Successful introduction of the vector into host cells can be monitored using various known methods. For example, transient translocation can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable translocation into cells in vitro can be ensured by using markers that provide the translocated cells with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0300] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentivirus vectors and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, including vaccinia virus vectors, or avipox, including canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may optionally include a viral sequence for translocation. Alternatively, the construct may be incorporated into an episomal replication vector, such as EPV and EBV vectors. Constructs for the recombinant expression of iRNAs generally require regulatory factors, such as promoters and enhancers, to ensure iRNA expression in target cells. Other aspects of vectors and constructs that are considered are described in more detail below.

[0301] A vector useful for delivering iRNA contains sufficient regulatory factors (promoters, enhancers, etc.) to express the iRNA in the desired target cells or tissues. These regulatory factors can be selected to provide either constitutive or regulatory / inducible expression.

[0302] iRNA expression can be precisely regulated using inductive regulatory sequences sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J.8:20-24). Suitable inductive expression systems for regulating dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, dimerizing chemical inducers, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0303] Viral vectors containing nucleic acid sequences encoding iRNA 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 the correct packaging of the viral genome and its integration into host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors to facilitate delivery of the nucleic acid to the patient. For more details on retroviral vectors, see 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 generate stem cells that exhibit higher resistance to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include 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 that may be considered include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; U.S. Patent No. 5,665,557; and U.S. Patent No. 5,981,276, which are incorporated herein by reference.

[0304] Adenoviruses are also being considered for use in iRNA delivery according to the present invention. Adenoviruses are particularly attractive vehicles for delivering genes, for example, to the airway epithelium. Adenoviruses infect the airway epithelium naturally, causing mild disease. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapies. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors for transferring genes into the airway epithelium 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 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); International Publication No. 94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing the iRNA addressed in this invention, a method for constructing a recombinant AV vector, and a method for delivering the vector to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0305] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNA of the present invention (Walsh et al., Proc.Soc.Exp.Biol.Med.204:289-300(1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNA may be expressed as two distinct complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNAs addressed in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described herein by reference in their entirety: 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 Publication No. 94 / 13788; and International Publication No. 93 / 24641.

[0306] Other viral vectors suitable for delivering the iRNA of the present invention include, for example, vaccinia viruses such as modified virus Ankara (MVA) or attenuated vaccinia such as NYVAC, and poxviruses such as avipox such as fowlpox or canarypox.

[0307] The affinity of a viral vector can be modified, if necessary, by pseudotyping the vector with coat proteins or other surface antigens from other viruses, or by substitution with capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mocola, etc. AAV vectors can be genetically engineered to target different cells by expressing different capsid protein serotypes; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, whose entire disclosure is incorporated herein by reference.

[0308] A vector-based drug may contain a vector in an acceptable diluent, or a sustained-release matrix in which a gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be generated intact from recombinant cells, the drug may contain one or more cells that generate the gene delivery system.

[0309] VI. Pharmaceutical composition of the present invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the present invention. In one embodiment, the present invention provides a pharmaceutical composition comprising the iRNA as described herein and a pharmaceutically acceptable carrier. The iRNA-containing pharmaceutical composition is useful for the treatment of diseases or disorders related to the expression or activity of the APOC3 gene. Such pharmaceutical compositions are formulated based on a delivery method. One example is a composition formulated for systemic administration via parenteral delivery, for example by subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, for example by injection into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the present invention may be administered in doses sufficient to inhibit the expression of the APOC3 gene. Generally, preferred doses of the iRNA of the present invention may range from about 0.001 to about 200.0 milligrams per kilogram of body weight per day of the recipient, generally ranging from about 1 to 50 mg per kilogram of body weight per day. For example, dsRNA can be administered in single doses of approximately 0.01 mg / kg, 0.05 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg.

[0310] For example, dsRNA is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, It may be administered in doses of 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0311] In another embodiment, dsRNA is approximately 0.1 to 50 mg / kg, approximately 0.25 to 50 mg / kg, approximately 0.5 to 50 mg / kg, approximately 0.75 to 50 mg / kg, approximately 1 to 50 mg / kg, approximately 1.5 to 50 mg / kg, approximately 2 to 50 mg / kg, approximately 2.5 to 50 mg / kg, approximately 3 to 50 mg / kg, approximately 3.5 to 50 mg / kg, approximately 4 to 50 mg / kg, approximately 4.5 to 50 mg / kg, approximately 5 to 50 mg / kg, approximately 7.5 to 50 mg / kg, approximately 10 to 50 mg / kg, approximately 15 to 50 mg / kg, approximately 20 to 50 mg / kg, approximately 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0. 5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, 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 kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30mg / kg, about 5 to about 30mg / kg, about 7.5 to about 30mg / kg, about 10 to about 30mg / kg, about 15 to about 30mg / kg, about 20 to about 30mg / kg, about 20 to about 30mg / kg, about 25 to about 30mg / kg, about 0.1 to about 20m It is administered in doses of g / kg, approximately 0.25–20 mg / kg, approximately 0.5–20 mg / kg, approximately 0.75–20 mg / kg, approximately 1–20 mg / kg, approximately 1.5–20 mg / kg, approximately 2–20 mg / kg, approximately 2.5–20 mg / kg, approximately 3–20 mg / kg, approximately 3.5–20 mg / kg, approximately 4–20 mg / kg, approximately 4.5–20 mg / kg, approximately 5–20 mg / kg, approximately 7.5–20 mg / kg, approximately 10–20 mg / kg, or approximately 15–20 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of this invention.

[0312] For example, dsRNA is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 It can be administered in doses of 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0313] In another embodiment, dsRNA is present in concentrations of approximately 0.5 to 50 mg / kg, 0.75 to 50 mg / kg, 1 to 50 mg / kg, 1.5 to 50 mg / kg, 2 to 50 mg / kg, 2.5 to 50 mg / kg, 3 to 50 mg / kg, 3.5 to 50 mg / kg, 4 to 50 mg / kg, 4.5 to 50 mg / kg, 5 to 50 mg / kg, 7.5 to 50 mg / kg, 10 to 50 mg / kg, 15 to 50 mg / kg, 20 to 50 mg / kg, 20 to 50 mg / kg, 25 to 50 mg / kg, and 25 to 50 mg / kg. g / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, 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 4 5mg / kg, about 20 to about 45mg / kg, about 25 to about 45mg / kg, about 25 to about 45mg / kg, about 30 to about 45mg / kg, about 35 to about 45mg / kg, about 40 to about 45mg / kg, about 0.5 to about 40mg / kg, about 0.75 to about 40mg / kg, about 1 to about 40mg / k g, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about Approximately 40 mg / kg, approximately 15 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 20 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 25 to approximately 40 mg / kg, approximately 30 to approximately 40 mg / kg, approximately 35 to approximately 40 mg / kg, approximately 0.5 to approximately 30 mg / kg, approximately 0.75 to approximately 30 m g / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.It is administered in doses of approximately 5-30 mg / kg, approximately 10-30 mg / kg, approximately 15-30 mg / kg, approximately 20-30 mg / kg, approximately 20-30 mg / kg, approximately 25-30 mg / kg, approximately 0.5-20 mg / kg, approximately 0.75-20 mg / kg, approximately 1-20 mg / kg, approximately 1.5-20 mg / kg, approximately 2-20 mg / kg, approximately 2.5-20 mg / kg, approximately 3-20 mg / kg, approximately 3.5-20 mg / kg, approximately 4-20 mg / kg, approximately 4.5-20 mg / kg, approximately 5-20 mg / kg, approximately 7.5-20 mg / kg, approximately 10-20 mg / kg, or approximately 15-20 mg / kg. In one embodiment, dsRNA is administered at a dose of approximately 10 mg / kg to approximately 30 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0314] For example, the target values ​​are approximately 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 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, 1 A single therapeutic dose of iRNA, such as 8, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 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 approximately 50 mg / kg, may be administered, for example, subcutaneously or intravenously. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0315] In some embodiments, the target includes doses of approximately 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0. 975, 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 0.3, 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, Multiple doses of therapeutic iRNA, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 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 approximately 50 mg / kg, are administered, for example, subcutaneously or intravenously. A multi-dose regimen may include daily administration of therapeutic iRNA over a period of 2, 3, 4, 5, 6, 7 days, or longer.

[0316] In other embodiments, the subjects include doses of approximately 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0. 975, 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 0.3, 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, Therapeutic doses of iRNA are administered in repeated doses, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 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 approximately 50 mg / kg, for example, subcutaneously or intravenously. Repeated dose regimens may include periodic administration of therapeutic doses of iRNA, such as every other day, every 3 days, every 4 days, twice a week, once a week, every other week, or once a month.

[0317] In certain embodiments, for example, when the composition of the present invention contains dsRNA and lipids as described herein, the target ranges are approximately 0.01 mg / kg to approximately 5 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.05 mg / kg to approximately 5 mg / kg, approximately 0.05 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, and approximately 0.2 mg / kg to approximately 5 mg / kg, about 0.2mg / kg to about 10mg / kg, about 0.3mg / kg to about 5mg / kg, about 0.3mg / kg to about 10mg / kg, about 0.4mg / kg to about 5mg / kg, about 0.4mg / kg to about 10mg / kg, about 0.5mg / kg to about 5mg / kg, about 0.5mg / kg to about 10mg / kg, about 1mg / kg to about 5mg / kg, about 1mg / kg to about 10mg / kg, about 1.5mg / kg to about 5mg / kg, about 1 .5mg / kg~about 10mg / kg, about 2mg / kg~about 2.5mg / kg, about 2mg / kg~about 10mg / kg, about 3mg / kg~about 5mg / kg, about 3mg / kg~about 10mg / kg, about 3.5mg / k g~about 5mg / kg, about 4mg / kg~about 5mg / kg, about 4.5mg / kg~about 5mg / kg, about 4mg / kg~about 10mg / kg, about 4.5mg / kg~about 10mg / kg, about 5mg / kg~about 10mg / Therapeutic doses of iRNA may be administered in amounts such as approximately 5.5 mg / kg to 10 mg / kg, 6 mg / kg to 10 mg / kg, 6.5 mg / kg to 10 mg / kg, 7 mg / kg to 10 mg / kg, 7.5 mg / kg to 10 mg / kg, 8 mg / kg to 10 mg / kg, 8.5 mg / kg to 10 mg / kg, 9 mg / kg to 10 mg / kg, or 9.5 mg / kg to 10 mg / kg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0318] For example, dsRNA is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5 It may be administered in doses of 0.2, 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. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of the present invention.

[0319] In certain embodiments of the present invention, for example, if the double-stranded RNAi agent includes modifications (e.g., one or more motifs of three identical modifications on a triple nucleotide sequence), for example, one such motif at or near the cleavage site of the agent, six phosphorothioate bonds, and a ligand, then such an agent may be present in doses of approximately 0.01 to approximately 0.5 mg / kg, approximately 0.01 to approximately 0.4 mg / kg, approximately 0.01 to approximately 0.3 mg / kg, approximately 0.01 to approximately 0.2 mg / kg, approximately 0.01 to approximately 0.1 mg / kg, approximately 0.01 mg / kg to approximately 0.09 mg / kg, and approximately 0.01 mg / kg to Approximately 0.08 mg / kg, approximately 0.01 mg / kg to approximately 0.07 mg / kg, approximately 0.01 mg / kg to approximately 0.06 mg / kg, approximately 0.01 mg / kg to approximately 0.05 mg / kg, approximately 0.02 to approximately 0.5 mg / kg, approximately 0.02 to approximately 0.4 mg / kg, approximately 0.02 to approximately 0.3 mg / kg, approximately 0.02 to approximately 0.2 mg / kg, approximately 0.02 to approximately 0.1 mg / kg, approximately 0.02 mg / kg to approximately 0.09 mg / kg, approximately 0.02 mg / kg to approximately 0.08 mg / kg, approximately 0.02 mg / kg to approximately 0.07 mg / kg, approximately 0.02 mg / kg to approximately 0.06 mg / kg, approximately 0 0.02mg / kg to approximately 0.05mg / kg, approximately 0.03 to approximately 0.5mg / kg, approximately 0.03 to approximately 0.4mg / kg, approximately 0.03 to approximately 0.3mg / kg, approximately 0.03 to approximately 0.2mg / kg, approximately 0.03 to approximately 0.1mg / kg, approximately 0.03mg / kg to approximately 0.09mg / kg, approximately 0.03mg / kg to approximately 0.08mg / kg, approximately 0.03mg / kg to approximately 0.07mg / kg, approximately 0.03mg / kg to approximately 0.06mg / kg, approximately 0.03mg / kg to approximately 0.05mg / kg, approximately 0.04 to approximately 0.5mg / kg, approximately 0.04 to approximately 0.4mg / kg, approximately 0.04 ~0.3mg / kg, 0.04~0.2mg / kg, 0.04~0.1mg / kg, 0.04mg / kg~0.09mg / kg, 0.04mg / kg~0.08mg / kg, 0.04mg / kg~0.07mg / kg, 0.04mg / kg~0. 0.06 mg / kg, about 0.05 to about 0.5 mg / kg, about 0.05 to about 0.4 mg / kg, about 0.05 to about 0.3 mg / kg, about 0.05 to about 0.2 mg / kg, about 0.05 to about 0.1 mg / kg, about 0.05 mg / kg to about 0.09 mg / kg, about 0.05 mg / kg to about 0.The drug is administered at a dose of 0.8 mg / kg, or approximately 0.05 mg / kg to approximately 0.07 mg / kg. Intermediate values ​​and ranges of the aforementioned enumerated values ​​are also intended to be part of the present invention; for example, RNAi agents may be administered to subjects at doses of approximately 0.015 mg / kg to approximately 0.45 mg / kg.

[0320] For example, RNAi agents, for example, RNAi agents in pharmaceutical compositions, are approximately 0.01 mg / kg, 0.0125 mg / kg, 0.015 mg / kg, 0.0175 mg / kg, 0.02 mg / kg, 0.0225 mg / kg, 0.025 mg / kg, 0.0275 mg / kg, 0.03 mg / kg, 0.0325 mg / kg, 0.035 mg / kg, 0.0 375mg / kg, 0.04mg / kg, 0.0425mg / kg, 0.045mg / kg, 0.0475mg / kg, 0.05mg / kg, 0.0525mg / kg, 0 .055mg / kg, 0.0575mg / kg, 0.06mg / kg, 0.0625mg / kg, 0.065mg / kg, 0.0675mg / kg, 0.07mg / kg, 0 .0725mg / kg, 0.075mg / kg, 0.0775mg / kg, 0.08mg / kg, 0.0825mg / kg, 0.085mg / kg, 0.0875mg / k g, 0.09mg / kg, 0.0925mg / kg, 0.095mg / kg, 0.0975mg / kg, 0.1mg / kg, 0.125mg / kg, 0.15mg / kg, 0 It may be administered in doses of 0.175 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, or about 0.5 mg / kg. Intermediate values ​​between the above-listed values ​​are also intended to be part of the present invention.

[0321] The pharmaceutical composition may be administered by intravenous infusion over a predetermined period of time, such as over 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or over approximately 25 minutes. Administration may be repeated regularly, such as weekly or bi-weekly (i.e., every two weeks), for a period of one, two, three, four months, or longer. After the initial treatment regimen, treatment may be administered at a lower frequency. For example, after weekly or bi-weekly administration for three months, administration may be repeated monthly for six months, one year, or longer.

[0322] The pharmaceutical composition may be administered once daily, or the iRNA may be administered in two or three or more partial doses at appropriate intervals throughout the day, or even by delivery via continuous infusion or controlled-release formulation. In this case, the amount of iRNA contained in each partial dose must be correspondingly smaller in order to achieve the total daily dose. The dosing unit may also be formulated for delivery over several days using conventional sustained-release formulations that provide sustained release of iRNA over several days, for example. Sustained-release formulations are well known in the art and are particularly useful for the delivery of the active ingredient to a specific site, which may be used with the active ingredient of the present invention. In this embodiment, the dosing unit contains a number of corresponding daily doses.

[0323] In another embodiment, a single dose of the pharmaceutical composition may be administered over a longer period, with subsequent doses given 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 present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered twice a month.

[0324] Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective dose of the composition may consist of a single treatment or a series of treatments. The effective doses and in vivo half-lives of individual iRNAs incorporated in this invention may be estimated using conventional procedures or based on in vivo studies using appropriate animal models, as described elsewhere in this specification.

[0325] The pharmaceutical compositions of the present invention may be administered in several ways, depending on whether topical or systemic treatment is desired and on the treatment area. Administration may be topical (e.g., by a transdermal patch), transpulmonary by inhalation or blowing of powder or fume, including by a nebulizer; intratracheal, intranasal, transepidermal and transdermal, oral or parenteral. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal administration via an implantable device, for example; or intracranial administration, such as within the brain parenchyma, subarachnoid space or ventricles.

[0326] iRNAs can be delivered in a manner that targets specific tissues, such as the liver (e.g., parenchymal cells of the liver).

[0327] 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, etc., may be necessary or desirable. Covered condoms, gloves, etc., may also be useful. Suitable topical formulations include those in which the iRNA addressed in this invention is in a miscible material with topically delivered substances such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNAs discussed in this invention can be encapsulated within liposomes or can form complexes with them, particularly with cationic liposomes. Alternatively, the iRNAs can form complexes with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaplate, tricaplate, monoolein, dilaurin, glyceryl 1-monocaplate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C1-20 alkyl esters (e.g., isopropylmyristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, incorporated herein by reference.

[0328] A. iRNA preparations containing membrane-like molecular assemblies The iRNAs used in the compositions and methods of the present invention may be formulated for delivery within membrane-like molecular assemblies, such as liposomes or micelles. In this specification, 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 monolayer or multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition but may optionally. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to a biological membrane, when liposomes are applied to tissue, the liposomal bilayer fuses with the cell membrane bilayer. As the fusion of the liposome and cell progresses, the internal aqueous contents containing the iRNA are delivered into the cell, where the iRNA can specifically bind to target RNA and mediate its delivery. In some cases, liposomes are also specifically targeted, for example, to induce iRNAs into specific cell types.

[0329] Liposomes containing iRNA agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed without the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid complex. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid, and lauroyl sarcosine. Next, the iRNA agent preparation is added to the micelles containing the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain the iRNA agent liposome preparation.

[0330] If necessary, a support compound, for example, that aids condensation can be added during the condensation reaction by controlled addition. For example, the support compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to further aid condensation.

[0331] A method for generating a stable polynucleotide delivery vehicle by incorporating a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle is further described, for example, in International Publication No. 96 / 37194, which is incorporated herein by reference in its entirety. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. 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. This may also include one or more embodiments of the exemplary methods described in al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing appropriately sized lipid aggregates for use as delivery vehicles include sonication and combinations of freeze-thaw and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). If consistently small (50–200 nm) and relatively uniform aggregates are desired, micro-solution preparation may be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable to the packing of RNAi preparations into liposomes.

[0332] Liposomes are broadly classified into two 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 taken into the endosome. Due to the acidic pH inside the endosome, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0333] pH-sensitive or negatively charged liposomes do not form complexes with nucleic acids; rather, they encapsulate them. Because nucleic acids and lipids both have similar charges, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used in culture to deliver nucleic acids encoding thymidine kinase genes to cell monolayers. Expression of exogenous genes was detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0334] One major type of liposome composition contains phospholipids in addition to naturally derived phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusion liposomes are mainly formed from dioleoyl sphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC. Yet another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0335] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; U.S. Patent Nos. 5,171,678; International Publication No. 94 / 00569; International Publication No. 93 / 24640; International Publication No. 91 / 16024; Felgner, 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.

[0336] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied, and their efficacy in drug delivery to the skin has been evaluated. Cyclosporine A was delivered into the dermis of mouse skin using nonionic liposome formulations containing Novasome® I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome® II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results suggested that such nonionic liposome systems are effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. STPPharma.Sci., 1994, 4, 6, 466).

[0337] Liposomes also include “stereostabilized” liposomes, which, as used herein, refer to liposomes containing one or more specialized lipids, which, when incorporated into liposomes, result in an improved cyclic lifespan compared to liposomes lacking such specialized lipids. Examples of stereostabilized liposomes include those in which a portion of the vesicle-forming lipid moiety of the liposome contains (A) one or more glycolipids such as monosialoganglioside GM1, or (B) one or more hydrophilic polymers such as polyethylene glycol (PEG) moiety. While we do not wish to be constrained by any particular theory, in the field of this technology, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivativeized lipids, the improvement in the circulating half-life of these sterically stabilized liposomes is thought to be due to reduced uptake by reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0338] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NYAcad. Sci., 1987, 507, 64) reported that monosialoganglioside GM1, galactocerebroside sulfate, and phosphatidylinositol can improve the blood half-life of liposomes. These findings were described in detail by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924, both granted to Allen et al., disclose liposomes containing (1) sphingomyelin and (2) ganglioside GM1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0339] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot efficiently fuse with the plasma membrane, but they can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0340] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; they can encapsulate a wide range of water- and lipid-soluble drugs; and they can protect iRNA agents encapsulated within their internal compartments from metabolism and degradation (Rosoff, “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Key considerations in the preparation of liposomal formulations include the lipid surface charge, vesicle size, and aqueous capacity of the liposomes.

[0341] Using the positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), small liposomes can be formed, which spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with negatively charged lipids in the cell membrane of tissue culture cells, resulting in iRNA delivery (for a description of DOTMA and its use in combination with DNA, see, for example, Felgner, Plet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355).

[0342] The ADOTMA analog 1,2-bis(oleoyloxy)-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 to cultured tissue cells, and it contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complex thus prepared spontaneously adheres to negatively charged cell surfaces, fuses with the plasma membrane, and efficiently delivers functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl portion is linked by an ester rather than an ether linkage.

[0343] Other reported cationic lipid compounds include those conjugated to a variety of moieties, such as carboxyspermine conjugated to one of two lipid types, and compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam®, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES") (see, for example, U.S. Patent No. 5,171,678).

[0344] Another cationic lipid complex involves lipid derivatization by cholesterol ("DC-Chol") combined with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for translocation 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 translocation than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California), and lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for oligonucleotide delivery are described in International Publication No. 98 / 39359 and International Publication No. 96 / 37194.

[0345] Liposome formulations are particularly well-suited for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver iRNA agents into the skin. In some implementations, liposomes are used to deliver iRNA agents to epidermal cells and to enhance the penetration of iRNA agents into skin tissues, such as within the skin. For example, liposomes can be applied topically. Topical delivery of therapeutic drugs formulated as liposomes to the skin has been demonstrated (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, R. Jand Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. Rand Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987.

[0346] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied, and their efficacy in drug delivery to the skin has been determined. Nonionic liposome 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 into the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0347] iRNA-containing liposomes can be highly deformable. Such deformability allows the liposomes to penetrate pores smaller than the average radius of the liposome. Transferosomes, for example, are a type of deformable liposome. Transferosomes can be created by adding surface edge activators, usually surfactants, to a standard liposome composition. Transferosomes containing iRNA agents can be delivered subcutaneously, for example by infection, to deliver the iRNA agent to keratinocytes in the skin. To cross intact mammalian skin, the lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of a suitable transcutaneous gradient. Furthermore, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and often self-load.

[0348] Other formulations to which the present invention can be applied are described in U.S. Provisional Patent Application No. 61 / 018,616, filed on 2 January 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed on 2 January 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed on 26 March 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed on 22 April 2008; and U.S. Provisional Patent Application No. 61 / 051,528, filed on 8 May 2008. PCT Application PCT / US2007 / 080331, filed on 3 October 2007, also describes formulations to which the present invention can be applied.

[0349] Transfersomes are yet another type of liposome, highly deformable lipid aggregates, that are attractive candidates for drug delivery vehicles. Because they are so highly deformable, transfersomes can be described as lipid droplets that can easily penetrate through pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to skin pore shapes), self-repair, and often reach and self-load their targets without fragmentation. 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 serum albumin-containing solution.

[0350] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and grading the properties of the many different types of surfactants, both natural and synthetic, is the use of the hydrophile / lipophile balance (HLB). The properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0351] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used across a wide pH range. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Examples of nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, also belong to this class. Polyoxyethylene surfactants are the most commonly found components of the nonionic surfactant class.

[0352] Surfactants are classified as anionic when their molecules retain a negative charge when dissolved or dispersed in water. Examples of anionic surfactants include carboxylates such as soap, acyl lactylate, acylamides of amino acids, sulfate esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0353] Surfactants are classified as cationic if their molecules retain a positive charge when dissolved or dispersed in water. Examples of cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used components in this class.

[0354] A surfactant is classified as amphoteric if its molecule has the ability to possess either a positive or negative charge. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phospholipids.

[0355] The use of surfactants in pharmaceuticals, formulations, and emulsions is outlined (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0356] The iRNA used in the method of the present invention may 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 such that all hydrophobic portions of the molecules face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. The opposite configuration exists if the environment is hydrophobic.

[0357] Mixed micelle formulations suitable for transdermal delivery may be prepared by mixing an siRNA composition, an alkali metal C8-C22 alkyl sulfate, and an aqueous solution of a micelle-forming compound. Examples of micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocolanyglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogues, polydocanol alkyl ether and its analogues, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles will form regardless of how the components are mixed, but they are mixed more vigorously to provide smaller micelles.

[0358] 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 an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by the addition of the remaining micelle-forming compounds with vigorous mixing.

[0359] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the preparation and protect it from bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonic agent such as glycerin may also be added after the mixed micelle composition has been formed.

[0360] To deliver a micelle formulation as a spray, the formulation can be placed in a fumigant metering and dispensing device, and the spray can be loaded into the device. Under pressurization, the spray is in liquid form within the metering and dispensing device. The ratio of components is adjusted so that the aqueous phase and the spray phase are one, i.e., a single phase. If there are two phases, the metering and dispensing device needs to be shaken, for example, before dispersing a portion of the contents through a metering valve. The dispensing dose of the pharmaceutical is sprayed in a fine mist from the metering valve.

[0361] Examples of spraying agents include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ethers, and diethyl ethers. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0362] The specific concentration of essential components can be determined by relatively simple experimental methods. For oral absorption, it is often desirable to increase the dose to, for example, at least two or three times the dose administered by injection or through the gastrointestinal tract.

[0363] B. Lipid particles The iRNA of the present invention, for example, dsRNA, may be completely encapsulated in a lipid preparation, such as LNP, or other nucleic acid-lipid particles.

[0364] As used herein, the term “LNP” refers to stable nucleic acid-lipid particles. LNPs typically include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs are extremely useful for systemic application because they exhibit a long circulating lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). Examples of LNPs include “pSPLP,” which contains encapsulation condensant-nucleic acid complexes as described in International Publication No. 00 / 03683. The particles of the present invention are substantially non-toxic and have an average diameter typically 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. In addition, when present in the nucleic acid-lipid particles of the present invention, the nucleic acids are resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent No. 5,976,567; U.S. Patent No. 5,981,501; U.S. Patent No. 6,534,484; U.S. Patent No. 6,586,410; U.S. Patent No. 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and International Publication No. 96 / 40964.

[0365] In one embodiment, the ratio (mass / mass ratio) of lipids to drugs (e.g., lipid to dsRNA ratio) is in the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1. Ranges between the ranges cited above are also considered to be part of the present invention.

[0366] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylamine. Minopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleyoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleythio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-Linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyoxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleyoyl-3-trimethylaminopropane chloride (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 (propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) The analogs thereof may be (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)butanoic acid (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazandiyl)didodecane-2-ol (Tech G1), or mixtures thereof. Cationic lipids can constitute approximately 20 mol% to 50 mol% or 40 mol% of the total lipids present in the particles.

[0367] 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.

[0368] In one embodiment, the lipid-siRNA particles consist of 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (molar percentage), with a particle size of 63.0 ± 20 nm and an siRNA / lipid ratio of 0.027.

[0369] Ionic / noncationic lipids include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerin (DOPG), dipalmitoyl phosphatidylglycerin (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine-4-(N-maleimide). These may include, but are not limited to, anionic or neutral lipids, such as 1-Cyl-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. If cholesterol is present, noncationic lipids may constitute about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0370] Conjugated lipids that inhibit particle aggregation may include, without limitation, polyethylene glycol (PEG) lipids, such as PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, and PEG-ceramide (Cer), or mixtures thereof. PEG-DAA complexes may include, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C8). The amount of conjugated lipids that prevent particle aggregation may be 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0371] In some embodiments, the nucleic acid-lipid particles further contain, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particles, which is cholesterol.

[0372] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be prepared using the lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, whose contents are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Each stock solution in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be combined in a molar ratio, for example, 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (for example, in sodium acetate at pH 5) so that the final ethanol concentration is about 35–45% and the final sodium acetate concentration is about 100–300 mM. Lipid-dsRNA nanoparticles typically form spontaneously during mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, such as a 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 replaced with phosphate-buffered saline (PBS) at approximately pH 7, such as approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. [ka]

[0373] The LNP01 formulation is described, for example, in International Publication No. 2008 / 042973 (which is incorporated herein by reference).

[0374] Further exemplary lipid-dsRNA preparations are listed in Table 1.

[0375] [Table 1]

[0376] [Table 2]

[0377] [Table 3]

[0378] Formulations comprising SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. 2009 / 127060, filed on April 15, 2009, which is incorporated herein by reference.

[0379] Formulations containing XTC are described, for example, in U.S. Provisional Application No. 61 / 148,366 filed on 29 January 2009; U.S. Provisional Application No. 61 / 156,851 filed on 2 March 2009; U.S. Provisional Application No. 1 filed on 10 June 2009; U.S. Provisional Application No. 61 / 228,373 filed on 24 July 2009; U.S. Provisional Application No. 61 / 239,686 filed on 3 September 2009; and International Application PCT / US2010 / 022614 filed on 29 January 2010.

[0380] Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed on June 10, 2010, which is incorporated herein by reference in its entirety.

[0381] Formulations containing ALNY-100 are described, for example, in the international application PCT / US 09 / 63933, filed on November 10, 2009, which is incorporated herein by reference.

[0382] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed on 5 May 2009, and International Application PCT / US10 / 33777, filed on 5 May 2010, which are incorporated herein by reference.

[0383] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, suspensions or solutions in water or aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. In some embodiments, the oral formulation is administered in combination with one or more osmotic 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, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, combinations of osmotic enhancers are used, such as fatty acid / salt combined with bile acid / salt. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA addressed in this invention may be delivered orally in granular form, including spray-dried particles, or may be complexed to form micro or nanoparticles.Examples of DsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pullulans, 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(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexyl Examples include acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, methyl polyacrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparations are described in detail in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Patent No. 6,747,014, respectively, which are incorporated herein by reference.

[0384] Compositions and formulations for parenteral, intracerebral (intracerebral), subarachnoid, intraventricular, or intrahepatic administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including but not limited to osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0385] Examples of the pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but are not limited to, pre-made liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver disorders such as liver cancer, formulations targeting the liver are particularly preferred.

[0386] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage forms, can be prepared according to the prior art well known in the pharmaceutical industry. Such art involves the step of combining the active ingredient with a pharmaceutical carrier or excipient. Generally, formulations are prepared by uniformly and closely combining the active ingredient with a liquid carrier or an ultrafine particle solid carrier or both, and then shaping the product if necessary.

[0387] The compositions of the present invention can be formulated into any of a number of possible dosage forms, including but not limited to tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspensions may also contain stabilizers.

[0388] C. Additional formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid, usually in the form of droplets with a diameter greater than 0.1 μm (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, 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., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301. Emulsions are often biphasic systems containing two immiscible liquid phases that are closely mixed and dispersed from one another. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed in a bulk oily phase as microdroplets, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed in a bulk aqueous phase as microdroplets, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to a dispersed phase and an active agent, which may exist as a solution in either an aqueous or oily 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 can also be multi-phase emulsions, such as oil-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer specific advantages that simple two-component emulsions do not. Among these, a multi-phase emulsion in which individual oil droplets of an o / w emulsion surround smaller water droplets constitutes a w / o / w emulsion. Similarly, an oil droplet system encapsulated in small water spheres and stabilized within a continuous oily phase provides an o / w / o emulsion.

[0389] Emulsions are characterized by having little to no thermodynamic stability. Often, the dispersed or discontinuous phases of an emulsion are well dispersed externally or within the continuous phase and maintained in this form through emulsifiers or means of increasing the formulation viscosity. In the case of emulsion-type ointment bases and creams, any of the emulsion phases may be semi-solid or solid. Another means of stabilizing an emulsion involves the use of emulsifiers, which may be incorporated into any of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorbent bases, and finely dispersed solids (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0390] Synthetic surfactants, also known as surfactants, have a wide range of applications in emulsion formulations and are outlined in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199). Surfactants are typically amphiphilic, containing both hydrophilic and hydrophobic moieties. The ratio of hydrophilic to hydrophobic groups is called the hydrophilic / lipophilic balance (HLB) of a surfactant and is a useful means of classifying and selecting surfactants in the preparation of pharmaceutical formulations. Surfactants can be classified into different classes based on the properties of their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0391] Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorbent bases with hydrophilic properties that can absorb water and form w / o emulsions, such as anhydrous lanolin and hydrophilic petrolatum, still maintain their semi-solid viscosity. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, particularly in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-expanding clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloids of aluminum silicate and magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0392] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199).

[0393] Examples of hydrophilic colloids include natural 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., carbomer, cellulose ether, and carboxyvinyl polymer). These disperse in water or swell in water to form a colloidal solution that stabilizes the emulsion by forming a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the outer phase.

[0394] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can readily support microbial growth; therefore, preservatives are frequently incorporated into these formulations. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene; reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0395] The application of emulsion formulations via cutaneous, oral, and parenteral routes, and methods for manufacturing them, are outlined in the literature. (See, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are widely used due to their ease of preparation and efficiency in terms of absorption and bioavailability (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199). Mineral oil-based laxatives, fat-soluble vitamins, and high-fat nutritional supplements are among the materials commonly administered orally as o / w emulsions.

[0396] ii. Microemulsion In one embodiment of the present invention, the iRNA and nucleic acid composition is prepared as a microemulsion. A microemulsion can be defined as a single optically isotropic and thermodynamically stable solution of water, oil, and an amphiphilic substance (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, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, a microemulsion is a system prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, which is generally an alcohol of intermediate chain length, to form a clear system. Therefore, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surfactant molecules (Leung and Shah, Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are typically prepared through a combination of 3 to 5 components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used, as well as the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.271).

[0397] Phenomenological approaches using phase diagrams have been extensively studied, providing those skilled in the art with comprehensive knowledge regarding the formulation of 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, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Block, 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 into spontaneously formed, thermodynamically stable droplet formulations.

[0398] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with co-surfactants. Typically, co-surfactants, which are short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant coating, resulting in an irregular coating due to gaps between 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 may, but is not limited to, water, aqueous solutions of pharmaceuticals, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0399] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as improved drug solubilization, protection of drugs from enzymatic hydrolysis, expected enhanced drug absorption due to changes in membrane fluidity and permeability induced by surfactants, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when compounding heat-unstable drugs, peptides, or iRNAs. Microemulsions have proven effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve local intracellular uptake of iRNAs and nucleic acids.

[0400] The microemulsion of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and enhance the absorption of the iRNA and nucleic acids of the present invention. Penetration enhancers used in the microemulsion of the present invention can be classified into 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.

[0401] iii. Particulates The iRNA agent of the present invention may be incorporated into particles, such as microparticles. Microparticles can be produced by spray drying, but they may also be produced by other methods, including freeze-drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0402] iv. Penetration enhancers In one embodiment, the present invention provides efficient delivery of nucleic acids, particularly iRNAs, to animal skin using various penetration enhancers. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been found that even non-lipophilic drugs can cross cell membranes if the membrane being traversed is treated with a penetration enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0403] Penetration enhancers can be classified into one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the aforementioned classes of penetration enhancers will be described in more detail below.

[0404] Surfactants (or "surface-activating agents") are chemical substances that, when dissolved in an aqueous solution, reduce the surface tension of the solution, or the interfacial tension between the aqueous solution and another liquid, thereby improving iRNA absorption through mucous membranes. In addition to bile salts and fatty acids, examples of these penetration enhancers include sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluoro compound emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0405] 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-monoleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, their C1-20 alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and di-glycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.). (See, for example, Touitou, E., et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0406] The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38; Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as osmotic enhancers. Therefore, the term “bile salt” includes any of the natural components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucoseic acid (sodium glucose), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE).(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, page 92; Swinyard, Chapter 39, 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).

[0407] Chelating agents used in connection with the present invention can be defined as compounds that remove metal ions from solution by forming complexes with them, thereby improving iRNA absorption through mucous membranes. With regard to their use as penetration enhancers in the present invention, since most DNA nucleases require divalent metal ions for catalytic activity and are inhibited by chelating agents, chelating substances have the additional advantage of also acting as deoxyribonuclease inhibitors (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating substances include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamine). (See, for example, Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0408] In the usage herein, non-chelating, non-surfactant osmotic enhancers can be defined as compounds that demonstrate insignificant activity as chelating agents or surfactants, but still enhance the absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of osmotic enhancers in this class include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0409] Substances that enhance iRNA uptake at the cellular level can also be added to the pharmaceuticals and other compositions of the present invention. For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188, granted to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (International Publication No. 97 / 30731, granted to Lollo et al.) are also known to enhance dsRNA uptake within cells. Examples of commercially available trait transfer reagents include, for example, Lipofectamine(trademark) (Invitrogen; Carlsbad, CA), Lipofectamine 2000(trademark) (Invitrogen; Carlsbad, CA), 293fectin(trademark) (Invitrogen; Carlsbad, CA), Cellfectin(trademark) (Invitrogen; Carlsbad, CA), DMRIE-C(trademark) (Invitrogen; Carlsbad, CA), FreeStyle(trademark)MAX(Invitrogen; Carlsbad, CA), and Lipofectamine(trademark) 2000. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), iRNAMAX(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), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec (trademark) / LipoGen (trademark) (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 (trademark) transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International;Examples include Mountain View, CA, USA, or HiFect® (trademark) (B-Bridge International, Mountain View, CA, USA).

[0410] Glycols such as ethylene glycol and propylene glycol; pyrroles such as 2-pyrrole; azone; and other active ingredients including terpenes such as limonene and menthone can be used to enhance the penetration of administered nucleic acids.

[0411] v. Carrier Certain compositions of the present invention also incorporate a carrier compound during formulation. In the use herein, “carrier compound” or “carrier” may refer to a nucleic acid or analogue that is inactive (i.e., not biologically active itself) but is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids, for example, by degrading biologically active nucleic acids or facilitating their removal from circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter, may result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory storage sites, possibly due to competition between the carrier compound and nucleic acid for the normal receptor. For example, the recovery of partial phosphorothioate dsRNAs in liver tissue may be reduced when administered concurrently with polyinosinate, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0412] vi. Excipients In contrast to carrier compounds, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected with the planned mode of administration in mind so as to provide the desired bulk, viscosity, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); bulking agents (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, silicon dioxide colloids, stearic acid, metal stearate salts, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0413] The compositions of the present invention can be prepared using pharmaceutically acceptable organic or inorganic excipients that do not react adversely with nucleic acids and are suitable for oral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0414] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil bases. The solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not cause adverse nucleic acid reactions and are suitable for oral administration may be used.

[0415] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0416] vii. Other ingredients The compositions of the present invention may further contain other auxiliary components found in conventional pharmaceutical compositions, at levels of use established in the art. Therefore, for example, the compositions may contain additional suitable pharmacologically active materials such as antipruritics, tannins, topical anesthetics, or anti-inflammatory agents, or additional materials useful for physically compounding the various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not excessively interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0417] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0418] In some embodiments, the pharmaceutical compositions covered by the present invention include (a) one or more iRNA compounds, and (b) one or more agents that function by a non-iRNA mechanism and are useful for treating APOC-related disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-lipid agents, antivirals, and / or anti-fibrotic agents. In addition, other substances commonly used to protect the liver, such as silymarin, may also be used in combination with the iRNAs described herein. Other agents useful for treating liver diseases include terbivudine, entecavir, telaprevir, and protease inhibitors such as those disclosed in U.S. Patent Application Publication 2005 / 0148548, U.S. Patent Application Publication 2004 / 0167116, and U.S. Patent Application Publication 2003 / 0144217 granted to Tung et al., and U.S. Patent Application Publication 2004 / 0127488 granted to Hale et al.

[0419] The toxicity and therapeutic effects of such compounds can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals to determine the LD50 (lethal dose in 50% of the population) and ED50 (therapeutably effective dose in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a high therapeutic index are preferred.

[0420] Data obtained from cell culture assays and animal experiments can be used to formulate dosage ranges for human use. Doses of the compositions discussed in this invention are generally within the range of circulating concentrations, including the ED50, which is minimally or completely toxic. Doses may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods discussed in this invention, the therapeutically effective dose can first be estimated from a cell culture assay. Doses may be formulated in animal models to achieve the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence, including the IC50 (i.e., the test compound concentration that achieves maximum half-dose inhibition of symptoms) determined in cell culture (e.g., achieving a reduction in polypeptide concentration). Such information can be used to more accurately determine useful doses in humans. Plasma...

Claims

1. A double-stranded RNAi agent for inhibiting the expression of apolipoprotein C3 (APOC3) in cells, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from the nucleotide sequence of SEQ ID NO:

2. Substantially all of the nucleotides in at least one strand are modified nucleotides, and A double-stranded RNAi agent in which the sense strand is coupled to a ligand attached to its 3' end.

2. The double-stranded RNAi agent according to claim 1, wherein the sense strand and the antisense strand include a complementary region comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the sequences listed in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, and 12, 13.

3. At least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-fixed nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramido salt, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, or a 1,5-anhydrohexitol modified nucleotide. A double-stranded RNAi agent according to claim 1 or 2, selected from the group consisting of creotide, cyclohexenyl-modified nucleotide, nucleotide containing a 5'-phosphorothioate group, nucleotide containing a 5'-methylphosphonate group, nucleotide containing 5'-phosphate or a 5'-phosphate mimetic, nucleotide containing vinyl phosphate, nucleotide containing adenosine-glycol nucleic acid (GNA), nucleotide containing a thymidine-glycol nucleic acid (GNA) S-isomer, nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide containing 2'-deoxythymidine-3'-phosphate, nucleotide containing 2'-deoxyguanosine-3'-phosphate, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoate bisdecylamide group.

4. The double-stranded RNAi agent according to claim 1, wherein substantially all of the nucleotides of the sense strand are modified.

5. The double-stranded RNAi agent according to claim 1, wherein substantially all of the nucleotides of the antisense strand are modified.

6. The double-stranded RNAi agent according to claim 1, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

7. The double-stranded RNAi agent according to claim 1, wherein all of the nucleotides in the sense strand are modified nucleotides.

8. The double-stranded RNAi agent according to claim 1, wherein all nucleotides of the antisense strand are modified nucleotides.

9. The double-stranded RNAi agent according to claim 1, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

10. The double-stranded RNAi agent according to claim 1, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

11. The double-stranded RNAi agent according to claim 1, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

12. A double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprises a region complementary to a part of the mRNA encoding APOC3, and each strand is approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is of formula (III): Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3' Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5'(III) [In the formula: i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; Each Na and Na' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides or a combination thereof, which is either modified or unmodified, and each sequence contains at least two different modified nucleotides; Each Nb and Nb' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, or a combination thereof, which is either modified or unmodified; Each np, np', nq, and nq' (each of which may or may not be present) independently represents an overhang nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on a triple nucleotide; The modifications on Nb are different from the modifications on Y, and the modifications on Nb' are different from the modifications on Y'; and The sense strand is coupled to at least one ligand in the double-stranded RNAi agent.

13. The double-stranded RNAi agent according to claim 12, 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.

14. The double-stranded RNAi agent according to claim 12, wherein k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1.

15. The double-stranded RNAi agent according to claim 12, wherein the YYY motif is present at or near the cleavage site of the sense strand.

16. The double-stranded RNAi agent according to claim 12, wherein the Y'Y'Y' motif is located at positions 11, 12, and 13 from the 5' end of the antisense strand.

17. The double-stranded RNAi agent according to claim 16, wherein Y' is 2'-O-methyl or 2'-fluoro.

18. Equation (III) is Equation (IIIa): Sense: 5'np-Na-YYY-Na-nq3' Antisense: 3'np'-Na'-Y'Y'Y'-Na'-nq'5'(IIIa) A double-stranded RNAi agent according to claim 12, as represented by [the specified figure].

19. The double-stranded RNAi agent according to claim 1 or 12, wherein the double-stranded region is 15 to 30 nucleotide pairs long.

20. The double-stranded RNAi agent according to claim 19, wherein the double-stranded region is 17 to 23 nucleotide pairs long.

21. The double-stranded RNAi agent according to claim 19, wherein the double-stranded region is 17 to 25 nucleotide pairs long.

22. The double-stranded RNAi agent according to claim 19, wherein the double-stranded region is 23 to 27 nucleotide pairs long.

23. The double-stranded RNAi agent according to claim 19, wherein the double-stranded region is 19 to 21 nucleotide pairs long.

24. The double-stranded RNAi agent according to claim 19, wherein the double-stranded region is 21 to 23 nucleotide pairs long.

25. A double-stranded RNAi agent according to claim 1 or 12, wherein each strand has 15 to 30 nucleotides.

26. A double-stranded RNAi agent according to claim 1 or 12, wherein each strand has 19 to 30 nucleotides.

27. The double-stranded RNAi agent according to claim 12, wherein the modification on the nucleotide is selected from the group consisting of the modifications listed in Tables 5, 9, 10, 11B, 12, and 13, and combinations thereof.

28. The double-stranded RNAi agent according to claim 12, wherein the modifications on the nucleotide are 2'-O-methyl and 2'-fluoro modifications.

29. The double-stranded RNAi agent according to claim 1 or 12, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

30. The ligand is 【Chemistry 1】 The double-stranded RNAi agent according to claim 1 or 12.

31. The double-stranded RNAi agent according to claim 1 or 12, wherein the ligand is attached to the 3' end of the sense strand.

32. The following schematic diagram 【Chemistry 2】 The double-stranded RNAi agent according to claim 31, which is conjugated with a ligand as shown in the formula [wherein X is O or S].

33. A double-stranded RNAi agent according to claim 1 or 12, further comprising at least one phosphorothioate or methylphosphonate internucleotide bond.

34. The double-stranded RNAi agent according to claim 33, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 3' end of one of the strands.

35. The double-stranded RNAi agent according to claim 34, wherein the strand is the antisense strand.

36. The double-stranded RNAi agent according to claim 34, wherein the strand is the sense strand.

37. The double-stranded RNAi agent according to claim 33, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 5' end of one of the strands.

38. The double-stranded RNAi agent according to claim 37, wherein the strand is the antisense strand.

39. The double-stranded RNAi agent according to claim 37, wherein the strand is the sense strand.

40. The double-stranded RNAi agent according to claim 33, wherein the phosphorothioate or methylphosphonate internucleotide bond is present at both the 5' and 3' ends of one of the strands.

41. The double-stranded RNAi agent according to claim 40, wherein the strand is the antisense strand.

42. The double-stranded RNAi agent according to claim 33, comprising 6 to 8 phosphorothioate nucleotide interbondings.

43. The double-stranded RNAi according to claim 42, wherein the antisense strand includes two phosphorothioate nucleotide interbonds at its 5' end and two phosphorothioate nucleotide interbonds at its 3' end, and the sense strand includes at least two phosphorothioate nucleotide interbonds at either its 5' or 3' end.

44. The double-stranded RNAi agent according to claim 1 or 12, wherein the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair.

45. The double-stranded RNAi agent according to claim 9, wherein the Y nucleotide contains a 2'-fluoro modification.

46. The double-stranded RNAi agent according to claim 12, wherein the Y' nucleotide contains a 2'-O-methyl modification.

47. The double-stranded RNAi agent according to claim 1 or 12, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

48. A double-stranded RNAi agent according to claim 1 or 12, selected from the group of RNAi agents listed in any one of Tables 4A, 4B, 5, 8, 9, 11A, 11B, 12, and 13.

49. A double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, The sense chain comprises 5'-GCUUAAAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-AGAAUACUGUCCCCUUUUAAGCAA-3' (SEQ ID NO: 14), Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides. The sense chain is coupled to the ligand attached to its 3' end, and The ligand is a double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

50. A double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, The sense chain comprises 5'-GCUUAAAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 13), and the antisense chain comprises 5'-UGAAUACUGUCCCCUUUUAAGCAA-3' (SEQ ID NO: 15), Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides. The sense chain is coupled to the ligand attached to its 3' end, and The ligand is a double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

51. A double-stranded RNAi agent having the ability to inhibit the expression of apolipoprotein C3 (APOC3) in cells, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, The sense strand comprises 5'-GCUUAAAAAAGGGACAGUAUUCA-3' (SEQ ID NO: 659), and the antisense strand comprises 5'-UGAAUACUGUCCCCUUUUAAGCAA-3' (SEQ ID NO: 670), Substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides. The sense chain is coupled to the ligand attached to its 3' end, and The ligand is a double-stranded RNAi agent, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

52. A double-stranded RNAi agent according to any one of claims 49 to 51, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified.

53. At least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-fixed nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramido salt, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, or a 1,5-anhydrohexitol modified nucleotide. A double-stranded RNAi agent according to any one of claims 49 to 51, selected from the group consisting of nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing 5'-phosphate or a 5'-phosphate mimetic, nucleotides containing vinyl phosphate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoate bisdecylamide group.

54. A double-stranded RNAi agent according to any one of claims 49 to 51, comprising 10 or fewer nucleotides containing 2'-fluoromodifications.

55. A double-stranded RNAi agent according to any one of claims 49 to 51, comprising six or fewer nucleotides containing 2'-fluoromodifications.

56. The double-stranded RNAi agent according to any one of claims 49 to 51, wherein the sense strand contains four or fewer nucleotides including 2'-fluoro modifications.

57. The double-stranded RNAi agent according to any one of claims 49 to 51, wherein the antisense strand contains six or fewer nucleotides including 2'-fluoromodifications.

58. The double-stranded RNAi agent according to any one of claims 49 to 51, wherein the antisense strand contains two or fewer nucleotides including a 2'-fluoro modification.

59. The double-stranded RNAi agent according to any one of claims 49 to 51, further comprising a 5'-phosphate or a 5'-phosphate mimetic at the 5' nucleotide of the antisense strand.

60. The double-stranded RNAi agent according to any one of claims 49 to 51, further comprising a 5'-phosphate mimetic in the 5' nucleotide of the antisense strand.

61. The double-stranded RNAi agent according to claim 60, wherein the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

62. The ligand is 【Transformation 3】 The double-stranded RNAi agent according to any one of claims 49 to 51.

63. The following schematic diagram 【Chemistry 4】 A double-stranded RNAi agent according to claim 62, which is conjugated with a ligand as shown in the formula [wherein X is O or S].

64. A double-stranded RNAi agent containing an RNAi sequence listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13.

65. The following array: Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3' (Sequence ID 16) Antisense: 5'asGfsaAfuAfcUfgUfccccUfuUfuAfaGfcsAfsa 3' (Sequence ID 17) The double-stranded RNAi agent according to claim 64, which is AD-57553 containing the above.

66. The following array: Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3' (Sequence ID 18) Antisense: 5'VPusGfsaAfuAfcUfgUfccccUfuUfuAfaGfcsasa 3' (Sequence ID 19) The double-stranded RNAi agent according to claim 64, which is AD-65696 containing the above.

67. The following array: Sense: 5'gscsuuaaAfaGfGfGfacaguauucaL96 3' (Sequence ID 20) Antisense: 5'usGfsaauAfcUfGfuccccUfuUfuaagcsasa 3' (Sequence ID 21) The double-stranded RNAi agent according to claim 64, which is AD-65703 containing the above.

68. The following array: Sense: 5'gscsuuaaAfaGfGfGfacaguauucaL96 3' (Sequence ID 22) Antisense: 5'usGfsaauacuguccccUfuuuaagcsasa 3' (Sequence ID 23) The double-stranded RNAi agent according to claim 64, which is AD-65704 containing the above.

69. The following array: Sense: 5'cscscaauAfaAfGfCfuggacaagaaL96 3' (Sequence ID 714) Antisense: 5'usUfscuuGfuuCfCfagcuuUfuuAfuugggsasag 3' (Sequence ID 718) The double-stranded RNAi agent according to claim 64, which is AD-67221 containing the above.

70. The following array: Sense: 5'gscsuuaaaaaGfgGfacaguaguauuca 3' (Sequence ID 738) Antisense: 5'sGfsaauacugucCfcUfuuuaagcsasa 3' (Sequence ID 749) The double-stranded RNAi agent according to claim 64, which is AD-69535 containing the above.

71. The following array: Sense: 5'gscsuuaaaaaGfgGfacagu(Agn)uuca 3'(Sequence ID 744) Antisense: 5'usGfsaauacugucCfcUfuuuaagcsasa 3' (Sequence ID 755) The double-stranded RNAi agent according to claim 64, which is AD-69541 containing the above.

72. A composition comprising a modified antisense polynucleotide agent, wherein the agent has the ability to inhibit APOC3 expression in cells and comprises a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, and the polynucleotide is about 14 to about 30 nucleotides long.

73. A vector containing a double-stranded RNAi agent according to any one of claims 1, 12, and 49 to 51.

74. Cells containing a double-stranded RNAi agent according to any one of claims 1, 12, and 49 to 51.

75. A composition comprising a double-stranded RNAi agent according to any one of claims 1, 12, and 49-51, or a modified antisense polynucleotide agent according to claim 72, or a pharmaceutical composition comprising a vector according to claim 73.

76. The pharmaceutical composition according to claim 75, wherein the double-stranded RNAi agent is present in a non-buffer.

77. The pharmaceutical composition according to claim 76, wherein the non-buffer solution is physiological saline or water.

78. The pharmaceutical composition according to claim 76, wherein the double-stranded RNAi agent is present in the buffer.

79. The pharmaceutical composition according to claim 78, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

80. The pharmaceutical composition according to claim 79, wherein the buffer solution is phosphate-buffered saline (PBS).

81. A method for inhibiting apolipoprotein C3 (APOC3) expression in cells, (a) The step of contacting the cells with a composition comprising a double-stranded RNAi agent according to any one of claims 1, 12, and 49-51, or a modified antisense polynucleotide agent according to claim 72, a vector according to claim 73, or a pharmaceutical composition according to any one of claims 75-80; and (b) A step of maintaining the cells produced in step (a) for a sufficient amount of time to achieve degradation of the mRNA transcript of the APOC3 gene, thereby inhibiting the expression of the APOC3 gene in the cells. A method that includes this.

82. The method according to claim 81, wherein the cells are located within the body of the subject.

83. The method according to claim 72, wherein the subject is a human or a rabbit.

84. The method according to claim 83, wherein the subject is suffering from APOC3-related disease.

85. The method according to any one of claims 81 to 84, wherein the APOC3 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

86. A method for treating a subject having an apolipoprotein C3 (APOC3) related disease, comprising the step of administering to the subject a therapeutically effective amount of a composition comprising a double-stranded RNAi agent according to any one of claims 1, 12, and 49 to 51, or a modified antisense polynucleotide agent according to claim 72, or a vector according to claim 73, or a pharmaceutical composition according to any one of claims 75 to 80, thereby treating the subject.

87. The method according to claim 86, wherein the APOC3-related disorder is hypertriglyceridemia.

88. The method according to claim 86, wherein the APOC3-related disease is selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance), hypertension, atherosclerosis, and pancreatitis.

89. The method according to claim 82, wherein the double-stranded RNAi agent is administered in a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg.

90. The method according to claim 89, wherein the double-stranded RNAi agent is administered in a dose of about 10 mg / kg to about 30 mg / kg.

91. The method according to claim 89, wherein the double-stranded RNAi agent is administered at a dose of approximately 3 mg / kg.

92. The method according to claim 89, wherein the double-stranded RNAi agent is administered at a dose of approximately 10 mg / kg.

93. The method according to claim 82, wherein the double-stranded RNAi agent is administered subcutaneously.

94. The method according to claim 82, wherein the double-stranded RNAi agent is administered intravenously.

95. The method according to claim 82, wherein the double-stranded RNAi agent is administered intramuscularly.

96. The method according to claim 82, wherein the RNAi agent is administered in two or more doses.

97. The method according to claim 95, wherein the RNAi agent is administered at intervals selected from the group consisting of once every 12 hours, once every 24 hours, once every 48 hours, once every 72 hours, and once every 96 hours.

98. The method according to claim 82, further comprising the step of administering an additional therapeutic agent to the subject.

99. The method according to claim 98, wherein the additional therapeutic agent is selected from the group consisting of HMG-CoA reductase inhibitors, fibrates, bile acid scavengers, niacin, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTTP) inhibitors, cholesterol modulators, bile acid modulators, peroxisome proliferation-activating receptor (PPAR) agonists, gene-based therapies, complex vascular protective agents, glycoprotein Ilb / IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors, squalene synthase inhibitors, monocyte chemotactic protein (MCP)-I inhibitors, or fish oil.

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