Sterol regulatory element-binding protein (SREBP) chaperone (SCAP) iRNA composition and method of use thereof

Abstract

The present invention provides compositions and methods for treating subjects with SCAP-related disorders. [Solution] The present invention provides a double-stranded ribonucleic acid (dsRNAi) agent and composition that targets the SCAP gene, as well as a method for inhibiting the expression of the SCAP gene using such a dsRNAi agent and composition, and a method for treating subjects with SCAP-related disorders such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 265,580, filed on 10 October 2015, and to U.S. Provisional Patent Application No. 62 / 378,964, filed on 24 August 2016. The aforementioned patent applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy, created on 8 December 2016, is named 121301-05020_SL.txt and has a size of 606,655 bytes. [Background technology]

[0003] The excessive accumulation of triglycerides in the liver is known as hepatic steatohepatosis (or fatty liver) and is associated with adverse metabolic consequences, including insulin resistance and dyslipidemia. Fatty liver is often found in individuals with excessive alcohol consumption and those with obesity, diabetes, or hyperlipidemia. However, non-alcoholic fatty liver disease (NAFLD) can develop even without excessive alcohol consumption (>10g / day). NAFLD refers to a broad range of liver diseases that can progress from simple fatty liver (steatohepatosis) to non-alcoholic steatohepatitis (NASH) and cirrhosis (irreversible, progressive scarring of the liver). All stages of NAFLD commonly involve the accumulation of fat (fatty infiltration) in liver cells (hepatocytes).

[0004] The NAFLD range begins with its simplest stage, called simple fatty liver (steatosis), and progresses from there. Simple fatty liver involves the accumulation of fat (triglycerides) in liver cells, without inflammation (hepatitis) or scarring (fibrosis). The next stage and severity in the NAFLD range is NASH, which involves inflammation of the liver along with the accumulation of fat in liver cells. Inflammatory cells destroy liver cells (hepatocyte necrosis), and NASH eventually leads to irreversible, progressive scarring (cirrhosis), after which scarring (fibrosis) of the liver occurs. Cirrhosis caused by NASH is the last and most severe stage in the NAFLD range.

[0005] NAFLD is now the leading cause of chronic liver disease in the United States, and there are currently about one million people diagnosed with NASH. Individuals with NASH have twice the risk of cardiovascular death compared to the general population, and NASH is the third most common cause of liver transplantation.

[0006] The central elements of lipid biosynthesis regulation in the human liver are a group of transcription factors called sterol regulatory element-binding proteins (SREBPs). There are three SREBP isoforms: SREBP-1a, SREBP-1c, and SREBP-2. In their precursor form, they are located in the endoplasmic reticulum (ER) (Non-Patent Literature 1; Non-Patent Literature 2) and, in the presence of cholesterol, bind to cholesterol and two other proteins: SCAP (SREBF chaperone) and Insig1 (insulin-inducible gene 1). When cholesterol levels decrease, Insig-1 dissociates from the SREBP-SCAP complex, allowing the complex to move to the Golgi apparatus, where SREBP is cleaved by two enzymes activated by SCAP, S1P and S2P (site 1 / 2 proteases; Non-Patent Literature 3; Non-Patent Literature 4). The cleaved SREBP then moves to the nucleus and acts as a transcription factor by binding to SREs (sterol regulatory elements) of several genes and stimulating their transcription (Non-Patent Literature 5). Among the genes that are transcribed are the LDL receptor, whose upregulation leads to increased cholesterol influx into the bloodstream; HMG-CoA reductase (Non-Patent Literature 6), the rate-limiting enzyme in de novo cholesterol synthesis; and several genes involved in fatty acid synthesis, such as Patatin-like phospholipase domain-containing gene 3 (PNPLA3), a multifunctional enzyme possessing both triacylglycerol lipase activity (triglyceride hydrolysis) and acylglycerol O-acyltransferase activity (triglyceride synthesis).

[0007] Since SCAP binding is essential for the transport and activation of all three SREBP isoforms, inhibiting SCAP expression and / or activity with agents that selectively and efficiently attenuate the body's own lipid biosynthesis, for example by inhibiting SCAP and thereby inhibiting SREBP activity, would be useful in treating pathological processes directly or indirectly mediated by SCAP expression. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Yokoyama C. et al.,Cell 1993,75:187 [Non-Patent Document 2] Hua X. et al.,Proc.Natl.Acad.Sci.1993,90:11603 [Non-Patent Document 3] Sakai J et al,Mol.Cell.1998,2:505 [Non-Patent Document 4] Rawson RBet al,Mol.Cell.1997,1:47 [Non-Patent Document 5] Briggs MRet al.,J.Biol.Chem.1993,268:14490 [Non-Patent Document 6] Anderson et al,Trends Cell Biol 2003,13:534 [Overview of the project] [Problems that the invention aims to solve]

[0009] Currently, treatment for NAFLD focuses on weight loss and the management of secondary conditions such as insulin resistance or dyslipidemia. However, no pharmacological treatments for NAFLD have been approved to date. Therefore, there is a need for treatment options for individuals with NAFLD. Consequently, there is a need for treatment options for individuals with NAFLD, such as dyslipidemia and NASH. [Means for solving the problem]

[0010] The present invention provides an iRNA composition that results in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of a sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene. The SCAP gene may be located within cells, for example, within cells in subjects such as humans. The present invention also provides a method for inhibiting the expression of the SCAP gene using the iRNA composition of the present invention, and / or a method for treating subjects who would benefit from the inhibition or reduction of SCAP gene expression, for example, subjects suffering from or susceptible to SCAP-related diseases, such as non-alcoholic fatty liver disease (NAFLD), such as non-alcoholic steatohepatitis (NASH).

[0011] Accordingly, in one embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of sterol regulatory element-binding protein (SREBP) chaperone (SCAP) genes. The double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 1 to 13, and the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 14 to 26.

[0012] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the SCAP gene, the double-stranded RNAi agent comprising a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ by three nucleotides or less from any one of the antisense sequences listed in any one of Tables 2, 3, 5, and 6.

[0013] In some embodiments, the sense strand and antisense strand of the double-stranded RNAi agent of the present invention include sequences selected from the group consisting of any one of the sequences in Tables 2, 3, 5, and 6.

[0014] In some embodiments, the sense strand is nucleotides 345-363, 378-396, 383-401, 402-420, 437-455, 458-476, 491-509, 1014-1032, 1237-1255, 1243-1261, 1259-1277, 1318-1336, 1323-1341, 1497-1515, 1571-1589, 1588-160 of SEQ ID NO: 2 6, 1605~1623, 1725~1743, 1767~1785, 1946~1964, 2004~2022, 2160~2178, 2193~2211, 2217~2235, 2517~2535, 2547~2565, 2616~2634, 2663~2681, 2717~2735, 2734~2752, 2751~2769, 2874~2892, 2885~2903, 2998~3016, 3276~3294, 3292~3310, 3325~3343, 3342~3360, 3420~3438, 3469~3487, 3478~3496, 3533~3551, 3549~3567, 3565~3583, 3579~3597, 3622~3640, 3667~3685, 3771~3789, 3788~3806, 3871~3889, 38 It includes the nucleotide sequences 91-3909, 3904-3922, 3921-3939, 4002-4020, 4010-4028, 4027-4045, 4075-4093, 4129-4147, 4145-4163, 4149-4167, 4168-4186, 4184-4202, and 4197-4215, and at least 15 consecutive nucleotides that differ by three nucleotides or less.

[0015] In some embodiments, the antisense chains are AD-77633, AD-77631, AD-77630, AD-77629, AD-77627, AD-77625, AD-77624, AD-77587, AD-77573, AD-77572, AD-77571, AD-77567, AD-77566, AD-77738, AD-77 731, AD-77730, AD-77729, AD-77721, AD-77718, AD-77706, AD-77701, AD-77690, AD-77688, AD-7768 6, AD-77669, AD-77667, AD-77662, AD-77660, AD-77656, AD-77655, AD-77654, AD-77555, AD-77554, A D-77547, AD-77530, AD-77529, AD-77527, AD-77526, AD-77521, AD-77519, AD-77518, AD-77514, AD- 77513, AD-77512, AD-77510, AD-77507, AD-77505, AD-77499, AD-77498, AD-77494, AD-77492, AD-774 It comprises a double-stranded antisense nucleotide sequence selected from the group consisting of 91, AD-77490, AD-77486, AD-77485, AD-77484, AD-77483, AD-77479, AD-77478, AD-77477, AD-77476, AD-77475, and AD-77474, and at least 15 consecutive nucleotides that differ by three nucleotides or less.

[0016] In certain embodiments, the double-stranded RNAi agent contains at least one modified nucleotide. In certain embodiments, the double-stranded RNAi agent contains four or fewer (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in the sense strand. In certain embodiments, the double-stranded RNAi agent contains four or fewer (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in the antisense strand. In certain embodiments, the dsRNA agent contains four or fewer (i.e., 4, 3, 2, 1, or 0) unmodified nucleotides in both the sense and antisense strands. In certain embodiments, substantially all of the nucleotides in the sense strand of the double-stranded RNAi agent are modified nucleotides. In certain embodiments, substantially all of the nucleotides in the antisense strand of the double-stranded RNAi agent are modified nucleotides. In certain embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand of the double-stranded RNAi agent are modified nucleotides.

[0017] In certain embodiments, the modified nucleotide is independently selected from the group consisting of 2'-O-methyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0018] In one embodiment, the modified nucleotides include deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally fixed nucleotides, restricted ethyl nucleotides, debasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, and 1,5-anhydrohexitol. The group consists of modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 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 cholesteryl derivatives and terminal nucleotides linked to a dodecanoate bisdecylamide group.

[0019] In certain embodiments, the modified nucleotide is selected from the group consisting of nucleotides including 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases. In some embodiments, the modified nucleotide includes a short sequence of a 3'-terminal deoxythymine nucleotide (dT). In certain embodiments, the nucleotide modifications are 2'-O-methyl modification and 2'-fluoro modification.

[0020] In some embodiments, the double-stranded RNAi agent further comprises at least one phosphorothioate nucleotide interbonding. In some embodiments, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate nucleotide interbondings, for example, 6, 7, or 8 phosphorothioate nucleotide interbondings.

[0021] In certain embodiments, the double-stranded RNAi agent includes a complementary region of at least 17 nucleotides in length. In certain embodiments, the double-stranded RNAi agent includes complementary regions of 19 and 23 nucleotides in length. In certain embodiments, the double-stranded RNAi agent includes a complementary region of 19 nucleotides in length.

[0022] In certain embodiments, each strand of the double-stranded RNAi agent is 30 nucleotides or less in length. In certain embodiments, the double-stranded RNAi agent is at least 15 nucleotides in length.

[0023] In certain embodiments, at least one strand of the double-stranded RNAi agent contains a 3' overhang of at least one nucleotide. In certain embodiments, at least one strand contains a 3' overhang of at least two nucleotides.

[0024] In some embodiments, the double-stranded RNAi agent further comprises a ligand. In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi agent. In one embodiment, the ligand is conjugated to the 3' end of the sense strand via a monovalent or branched divalent or trivalent linker. In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative conjugated to the 3' end of the sense strand of the double-stranded RNAi agent. In certain embodiments, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi agent via a branched divalent or trivalent linker. In certain embodiments, the ligand is [ka] That is the case.

[0025] In certain embodiments, the double-stranded RNAi agent is conjugated to a ligand as shown in the following schematic diagram [Chemical formula] where X is O or S.

[0026] In some embodiments, X is O. In certain embodiments, the ligand is cholesterol.

[0027] In some embodiments, the complementary region comprises any one of the antisense sequences in any one of Tables 2, 3, 5, and 6. In other embodiments, the complementary region consists of any one of the antisense sequences in any one of Tables 2, 3, 5, and 6.

[0028] In one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of a sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’ (III) (In the formula, i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is independently either modified or unmodified, or a combination thereof; each n p , n p ',n q , and n q ' represents an overhanging nucleotide, which may or may not be present; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications to three consecutive nucleotides; N b The modification to N is different from the modification to Y, and N b Modifications to ' are represented by (which are different from modifications to Y'); the sense chain is conjugated to at least one ligand.

[0029] In some embodiments, 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 some embodiments, 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.

[0030] In some embodiments, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'. In some embodiments, the YYY motif is located at or near the cleavage site of the sense chain. In some embodiments, the Y'Y'Y' motif is located at positions 11, 12, and 13 from the 5' end of the antisense chain. In some embodiments, Y' is 2'-O-methyl.

[0031] In some embodiments, equation (III) is equation (IIIa): Sense: 5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5' (IIIa) It is represented by [this].

[0032] In some embodiments, equation (III) is equation (IIIb): Sense: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5' (IIIb) (In the formula, each N b and N b ' represents an oligonucleotide sequence containing 1 to 5 modified nucleotides independently.

[0033] In some embodiments, equation (III) is equation (IIIc): Sense: 5'n p -N a -XXX-N b -YYY-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5' (IIIb) (In the formula, each N b and N b' represents an oligonucleotide sequence containing 1 to 5 modified nucleotides independently.

[0034] In some embodiments, equation (III) is equation (IIId): Sense: 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5' (IIId) (In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N a and N a ' represents an oligonucleotide sequence containing 2 to 10 modified nucleotides independently.

[0035] In some embodiments, the double-stranded region is 15 to 30 nucleotide pairs long. In some embodiments, the double-stranded region is 17 to 23 nucleotide pairs long. In some embodiments, the double-stranded region is 17 to 25 nucleotide pairs long. In some embodiments, the double-stranded region is 23 to 27 nucleotide pairs long. In some embodiments, the double-stranded region is 19 to 21 nucleotide pairs long. In some embodiments, the double-stranded region is 21 to 23 nucleotide pairs long. In some embodiments, each strand has 15 to 30 nucleotides. In some embodiments, each strand has 19 to 30 nucleotides.

[0036] In some embodiments, the modification to the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In some embodiments, the modification to the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0037] In some embodiments, the ligand is one or more GalNAc derivatives or cholesterol linked via a divalent or trivalent branched linker.

[0038] In some embodiments, the ligand is [ka] That is the case.

[0039] In some embodiments, the ligand is bound to the 3' end of the sense strand. In some embodiments, the RNAi agent is shown in the schematic diagram below. [ka] It is conjugated to the ligand as shown.

[0040] In some embodiments, the drug further comprises at least one phosphorothioate or methylphosphonate internucleotide bond. In some embodiments, the phosphorothioate or methylphosphonate internucleotide bond is located at the 3' end of one of the strands. In some embodiments, this strand is an antisense strand. In some embodiments, this strand is a sense strand.

[0041] In some embodiments, the phosphorothioate or methylphosphonate internucleotide bond is located at the 5' end of one of the strands. In some embodiments, the strand is an antisense strand. In some embodiments, the strand is a sense strand.

[0042] In some embodiments, the phosphorothioate or methylphosphonate internucleotide bond is located at both the 5' and 3' ends of one of the strands. In some embodiments, the strand is an antisense strand.

[0043] In some embodiments, the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair. In some embodiments, the Y nucleotide contains a 2'-fluoro modification. In some embodiments, the Y' nucleotide contains a 2'-O-methyl modification. In some embodiments, p'>0. In some embodiments, p'=2. In some embodiments, q'=0, p=0, q=0, and the p' overhang nucleotide is complementary to the target mRNA. In some embodiments, q'=0, p=0, q=0, and the p' overhang nucleotide is not complementary to the target mRNA.

[0044] In some embodiments, the sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides. In some embodiments, at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond. In some embodiments, all n p ' is linked to an adjacent nucleotide via a phosphorothioate bond.

[0045] In some embodiments, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 2, 3, 5, and 6. In some embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified.

[0046] In another embodiment, the present invention provides cells containing a double-stranded RNAi agent provided herein.

[0047] In yet another aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of the SCAP gene. The composition comprises the double-stranded RNAi agent provided herein. In certain embodiments, the RNAi agent is administered in a non-buffered solution. In some embodiments, the non-buffered solution is saline or water. In other embodiments, the RNAi agent is administered by a buffered solution. In some embodiments, the buffered solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In some embodiments, the buffered solution is phosphate buffered saline (PBS). In some embodiments, the pharmaceutical composition further comprises a lipid formulation. In some embodiments, the lipid formulation comprises LNP. In some embodiments, the lipid formulation comprises MC3.

[0048] In one aspect, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, each strand is from about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’ (III) (wherein i, j, k, and l are each independently 0 or 1; p, p’, q, and q’ are each independently from 0 to 6; each N a and N a’ independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which can be either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ’ independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which can be either modified or unmodified or a combination thereof; each n p , n p ’, n q and n q ’ may or may not exist respectively and independently represent overhang nucleotides; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent one motif of three identical modifications for three consecutive nucleotides, and the modification is a 2’-O-methyl or 2’-fluoro modification; the modification for N b is different from the modification for Y, and the modification for N b ’ is different from the modification for Y’) is represented by; the sense strand is conjugated to at least one ligand.

[0049] In another aspect, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells. Here, the double-stranded RNAi agent contains a sense strand complementary to the antisense strand, and the antisense strand contains a region complementary to a part of the mRNA encoding SCAP. Each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b'-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n q , and n q ' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif of three identical modifications to three consecutive nucleotides, where these modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N b The modification to N is different from the modification to Y, and N b Modifications to ' are represented by (which are different from modifications to Y'); the sense chain is conjugated to at least one ligand.

[0050] In another embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p-N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n q , and n q ' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif of three identical modifications to three consecutive nucleotides, where these modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N b The modification to N is different from the modification to Y, and N bModifications to ' are represented by (different from modifications to Y'); the sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0051] In yet another embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n q , and n q ' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, which is either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif of three identical modifications to three consecutive nucleotides, where the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modification to N is different from the modification to Y, and N b Modifications to ' are represented by (which are different from modifications to Y'); the sense chain contains at least one phosphorothioate bond; the sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0052] In another embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p -N a -YYY-N a -n q 3' Antisense: 3'n p '-N a '-Y'Y'Y'-N a '-n q '5' (IIIa) (In the formula, each n p , n q , and n q' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a ' independently represents an oligonucleotide sequence containing 0-25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent a motif of three identical modifications to three consecutive nucleotides, represented by (the modifications being 2'-O-methyl or 2'-fluoro modifications); the sense strand contains at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0053] In one embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-nq '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is independently either modified or unmodified, or a combination thereof; each n p , n p ',n q , and n q Each of the following independently represents an overhanging nucleotide, which may or may not be present; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif of three identical modifications to three consecutive nucleotides, where these modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N b The modification to N is different from the modification to Y, and N b Modifications to ' are represented by (which are different from modifications to Y'); the sense chain is conjugated to at least one ligand.

[0054] In another embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a-n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n q , and n q ' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif of three identical modifications to three consecutive nucleotides, where these modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N b The modification to N is different from the modification to Y, and N b Modifications to ' are represented by (which are different from modifications to Y'); the sense chain is conjugated to at least one ligand.

[0055] In another embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n q , and n q ' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif of three identical modifications to three consecutive nucleotides, where these modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N b The modification to N is different from the modification to Y, and N b Modifications to ' are represented by (different from modifications to Y'); the sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0056] In yet another embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3 Antisense: 3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) (In the formula, i, j, k, and l are each independently 0 or 1; each n p , n q , and n q' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are independently modified, unmodified, or a combination thereof, and each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represents one motif of three identical modifications to three consecutive nucleotides, where these modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N b The modification to N is different from the modification to Y, and N b Modifications to ' are represented by (different from modifications to Y'); the sense chain contains at least one phosphorothioate bond; the sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0057] In another embodiment, the present invention provides a double-stranded RNAi agent that inhibits the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, wherein the double-stranded RNAi agent comprises an antisense strand and a sense strand complementary thereto, the antisense strand comprising a region complementary to a portion of the mRNA encoding SCAP, each strand being approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent is given by formula (III): Sense: 5'n p -N a -YYY-N a -n q 3' Antisense: 3'np '-N a '-Y'Y'Y'-N a '-n q '5' (IIIa) (In the formula, each n p , n q , and n q ' represents an overhang nucleotide, which may or may not be present; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds; each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent a motif of three identical modifications to three consecutive nucleotides, which are represented by 2'-O-methyl modifications or 2'-fluoro modifications; the sense strand contains at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, which is one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0058] In yet another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of sterol regulatory element-binding protein (SREBP) chaperone (SCAP) genes, 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 any one of the nucleotide sequences of SEQ ID NOs: 1 to 13, and the antisense strand comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs: 14 to 26, and the nucleotides of the sense strand Substantially all of the nucleotides in the sense strand include modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, the sense strand includes two phosphorothioate nucleotide interlinks at its 5' end, and substantially all of the nucleotides in the antisense strand include modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, 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 is conjugated to one or more GalNAc derivatives linked at its 3' end via a branched divalent or trivalent linker.

[0059] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of a sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene, 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 any one of the nucleotide sequences of SEQ ID NOs. 1 to 13, and the antisense strand comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 14 to 26, the sense strand comprising at least one 3'-terminal deoxythymine nucleotide (dT), and the antisense strand comprising at least one 3'-terminal deoxythymine nucleotide (dT).

[0060] In another embodiment, the present invention provides cells containing a double-stranded RNAi agent provided herein.

[0061] The present invention also provides a method for inhibiting SCAP expression in cells. This method comprises the steps of: contacting cells with a double-stranded RNAi agent or pharmaceutical composition as described herein; and maintaining the cells for a time sufficient to achieve degradation of the mRNA transcript of the SCAP gene, thereby inhibiting the expression of the SCAP gene in the cells.

[0062] In certain embodiments, the cells are located within the subject. In certain embodiments, the subject is a human being. In one embodiment, the subject is a human female. In another embodiment, the subject is a human male.

[0063] In certain embodiments, the human subject suffers from a SCAP-related disease. In one embodiment, the SCAP-related disease is non-alcoholic fatty liver disease (NAFLD). In another embodiment, the SCAP-related disease is fatty liver (steatosis). In yet another embodiment, the SCAP-related disease is non-alcoholic steatohepatitis (NASH). In yet another embodiment, the SCAP-related disease is obesity. In yet another embodiment, the SCAP-related disease is insulin resistance.

[0064] In a particular embodiment, SCAP expression is inhibited by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.

[0065] In another embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced SCAP expression. The method comprises the step of administering a therapeutically effective amount of any double-stranded RNAi agent or pharmaceutical composition provided herein to the subject, thereby treating the subject.

[0066] In certain embodiments, the human subject suffers from a SCAP-related disease. In one embodiment, the SCAP-related disease is non-alcoholic fatty liver disease (NAFLD). In another embodiment, the SCAP-related disease is fatty liver (steatosis). In yet another embodiment, the SCAP-related disease is non-alcoholic steatohepatitis (NASH). In yet another embodiment, the SCAP-related disease is obesity. In yet another embodiment, the SCAP-related disease is insulin resistance.

[0067] In certain embodiments, administration of this double-stranded RNAi agent to a subject results in a decrease in one or more serum lipids, such as triglycerides, and / or a decrease in SCAP protein accumulation. In certain embodiments, administration of this double-stranded RNAi agent to a subject results in a decrease in PNPLA3 protein accumulation or SREBP protein accumulation.

[0068] In certain embodiments, the method of the present invention further includes the administration of additional therapeutic agents and / or treatments, such as lifestyle modifications, e.g., exercise, weight loss, dietary changes, antioxidant therapy, omega-3 fatty acid intake, and / or liver transplantation.

[0069] In some embodiments, the double-stranded RNAi agent is administered at a dose of approximately 0.01 mg / kg to approximately 50 mg / kg. In other embodiments, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0070] In a further embodiment, the present invention also provides a method for inhibiting SCAP expression in a subject. This method comprises the step of administering a therapeutically effective amount of any of the double-stranded RNAi agents or pharmaceutical compositions provided herein to a subject, thereby inhibiting SCAP expression in the subject.

[0071] In another aspect, the present invention provides a method for reducing plasma triglyceride levels in a subject. The method includes administering to the subject a therapeutically effective amount of either the double-stranded RNAi agent or the pharmaceutical composition provided herein, thereby reducing the plasma triglyceride levels in the subject.

[0072] In another aspect, the present invention provides a method for inhibiting the progression of NAFLD in a subject, such as the progression from steatosis to NASH in a subject with steatosis or at risk of developing steatosis, e.g., a subject with insulin resistance or obesity; or the progression from NASH to cirrhosis in a subject with NASH or at risk of developing cirrhosis. The method includes administering to the subject a therapeutically effective amount of either the double-stranded RNAi agent or the pharmaceutical composition provided herein, thereby inhibiting the progression of NAFLD in the subject.

[0073] In some embodiments, the method of the present invention may further include determining the serum aspartate aminotransferase (AST) level in the subject, the alanine aminotransferase (ALT) level in the subject, the plasma triglyceride level in the subject, and / or determining the hepatic fat level in the subject.

[0074] In yet another aspect, the present invention provides a kit for performing the method of the present invention. In one embodiment, the present invention provides a kit for performing a method of inhibiting the expression of the SCAP gene in a cell by contacting the cell with an amount of the double-stranded RNAi agent of the present invention effective to inhibit the expression of SCAP in the cell. The kit includes the RNAi agent, instructions for use, and optionally, means for administering the RNAi agent to a subject.

Mode for Carrying Out the Invention

[0075] The present invention provides an iRNA composition that effects RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of a sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene. The SCAP gene can be intracellular, for example, within a cell in a subject such as a human. The present invention also provides a method of inhibiting the expression of the SCAP gene using the iRNA composition of the present invention, and / or a disorder that would benefit from inhibition or reduction of the expression of the SCAP gene, for example, an SCAP-related disease, for example, non-alcoholic fatty liver disease (NAFLD), for example, non-alcoholic steatohepatitis (NASH), and a method of treating a subject having the same.

[0076] Examples of the iRNA of the present invention include an RNA strand (antisense strand) having a region of about 30 nucleotide lengths or less, such as 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 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length, and the region is substantially complementary to at least a part of the mRNA transcript of the SCAP gene.

[0077] In certain embodiments, the iRNA of the present invention includes an RNA strand (antisense strand) that is longer in length, for example, up to 66 nucleotides, including regions of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of the C3 gene, for example, 36-66, 26-36, 25-36, 31-60, 22-43, and 27-53 nucleotides in length. These iRNAs having longer antisense strands preferably include a second RNA strand (sense strand) of 20-60 nucleotides in length, where the sense strand and antisense strand form a double helix of 18-30 consecutive nucleotides.

[0078] These iRNAs enable the targeted degradation of mammalian SCAP gene mRNA. In particular, extremely low doses of SCAP iRNA can specifically and efficiently mediate RNA interference (RNAi), potentially leading to significant inhibition of SCAP gene expression. Using cell-based assays, the inventors demonstrated that SCAP-targeting iRNAs can mediate RNAi and significantly inhibit SCAP gene expression. Therefore, methods and compositions containing these iRNAs are useful for treating subjects who would benefit from reduced levels and / or activity of the SCAP protein, such as those with SCAP-related diseases, e.g., non-alcoholic fatty liver disease (NAFLD), e.g., non-alcoholic steatohepatitis (NASH).

[0079] The following detailed description discloses methods for preparing and utilizing compositions containing iRNAs that inhibit the expression of the SCAP gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from the inhibition and / or reduction of the expression of this gene.

[0080] I. Definition To facilitate understanding of this invention, certain terms are defined first. In addition, it should be noted that whenever parameter values ​​or ranges are enumerated, intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of this invention.

[0081] 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, “factor” means one factor or two or more factors, such as multiple factors.

[0082] The term “including” is used herein to mean, and is used without distinction from, the term “including, but not limited to.” The term “or” is used herein to mean, and is used synonymously with, the term “and / or,” unless otherwise clearly indicated by the context.

[0083] The term “approximately” is used herein to mean within a range of typical errors in the art. For example, “approximately” can be understood as approximately 2 standard deviations from the mean. In certain embodiments, “approximately” means ±10%. In certain embodiments, “approximately” means ±5%. When “approximately” is placed before a set of numbers or ranges, it is understood that “approximately” can modify each of the numbers within the set of numbers or ranges.

[0084] The term “at least” placed before a number or a range of numbers is understood to include the number adjacent to the term “at least,” and any subsequent number or integer that can logically be included, as is evident from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides in a 21-nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the characteristic indicated. When “at least” is placed before a range of numbers or numbers, it is understood that “at least” can change any of the numbers in the above range or number of numbers.

[0085] In the usage of this specification, “less than or equal to” or “less than” is understood to mean a value or integer that is logically lower than the number adjacent to the phrase and, as logical from the context, is zero. For example, a double strand with an overhang of “less than or equal to 2 nucleotides” has an overhang of 2, 1, or 0 nucleotides. When “less than or equal to” is placed before a set of numbers or ranges, it is understood that “less than or equal to” can change any of the numbers within the set of numbers or ranges.

[0086] The term "SCAP" refers to a sterol regulatory element-binding protein (SREBP) chaperone having an amino acid sequence of any vertebrate or mammalian origin, including but not limited to humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs, also known as SREBP cleavage-activating protein, unless otherwise specified. The term also refers to fragments and variants of native SCAP that maintain at least one in vivo or in vitro activity of native SCAP. The term encompasses the full-length, unprocessed precursor form of SCAP, as well as the mature form resulting from post-translational cleavage of the signal peptide. The nucleotide and amino acid sequences of human SCAP can be found, for example, in GenBank accession number GI:66932901 (NM_012235.2; SEQ ID NO: 1).

[0087] The nucleotide and amino acid sequences of human SCAP are also, for example, GenBank accession number GI:987996597(NM_012235.3;SEQ ID NO:2);GenBank accession number GI:530372097(XM_005264967.1;SEQ ID NO:3);GenBank accession number GI:530372099(XM_005264968.1;SEQ ID NO:4);GenBank accession number GI:530372101(XM_005264969.1;SEQ ID NO:5);GenBank accession number GI: See also 767922691(XM_011533501.1; Sequence ID 6);GenBank accession number GI:767922693(XM_011533502.1; Sequence ID 7);GenBank accession number GI:767922696(XM_005264970.3; Sequence ID 8);GenBank accession number GI:530372105(XM_005264971.1; Sequence ID 9); and GenBank accession number GI:767922697(XM_005264972.3; Sequence ID 10).

[0088] The nucleotide and amino acid sequences of the cynomolgus monkey SCAP can be found, for example, in GenBank accession number GI:544413972 (XM_005546963.1; SEQ ID NO: 11). The nucleotide and amino acid sequences of the mouse SCAP can be found, for example, in GenBank accession number GI:557636668 (NM_001103162.2; SEQ ID NO: 12). The nucleotide and amino acid sequences of the rat SCAP can be found, for example, in GenBank accession number GI:155369286 (NM_001100966.1; SEQ ID NO: 13). Further examples of SCAP sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0089] The term “SCAP” also, as used herein, refers to certain polypeptides expressed in cells due to naturally occurring DNA sequence mutations in the SCAP gene, such as single nucleotide polymorphisms (SNPs) in the SCAP gene. Many SNPs have been identified within the SCAP gene; see, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov / snp). For a non-exclusive list of SNPs within the SCAP gene, see NCBI dbSNP accessions rs926103;rs12487736;rs12490383;rs754498;rs877097;rs878659;rs881264;rs900690;rs900691;rs900692;rs909200;rs909201;rs928391; or rs943555.

[0090] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the SCAP gene, including mRNA, which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence may be long enough to serve as a substrate for iRNA-directed cleavage in or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the SCAP gene.

[0091] 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 nucleotides in length. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0092] In the use of this specification, the term “sequence-containing chain” refers to an oligonucleotide containing a nucleotide chain described by the sequence mentioned, using standard nucleotide nomenclature.

[0093] "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 moieties, as further detailed below (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be replaced with other moieties without substantially altering the base-pairing properties of oligonucleotides containing such substituted moieties. As an example not intended to be limiting, a nucleotide containing inosine as a base can base-pair with a nucleotide containing adenine, cytosine, or uracil. 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, forming GU fluctuation base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods discussed in this invention.

[0094] 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. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, for example, inhibits the expression of the SCAP gene in cells, such as cells in mammalian subjects.

[0095] In one embodiment, the RNAi agent of the present invention is, for example, a single-stranded RNA that interacts with a target RNA sequence, such as a SCAP target mRNA sequence, to induce 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). Dicer, a ribonuclease-III-like enzyme, processes these dsRNAs into small interfering RNAs of 19-23 base pairs with a characteristic 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to induce 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). Thus, in one embodiment, the present invention relates to a single-stranded RNA (ssRNA) (the antisense strand of the siRNA double strand) that is generated in a cell to promote the formation of the RISC complex and result in the silencing of a target gene, namely the SCAP gene. Accordingly, the term "siRNA" is also used herein to refer to the RNAi as described above.

[0096] 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, Argonaute2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are incorporated herein by reference in their entireties from U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894. Any antisense nucleotide sequence described herein may be used as a single-stranded siRNA described herein or chemically modified by the method described in Lima et al., (2012) Cell 150;:883-894.

[0097] In another embodiment, the "iRNA" used in the compositions and methods of the present invention is double-stranded RNA, referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a ribonucleic acid molecule complex having a double-stranded structure that includes two anti-parallel and substantially complementary nucleic acid strands that are referred to as having "sense" and "antisense" orientations with respect to a target RNA, i.e., the SCAP gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) causes degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0098] 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 contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, in the use herein, “RNAi agent” may contain ribonucleotides having chemical modifications; an RNAi agent may contain substantial modifications to multiple nucleotides. In the use herein, the term “modified nucleotide” independently refers to a nucleotide 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 kinds of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are encompassed by “RNAi agent” for the purposes of this specification and the claims.

[0099] 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.

[0100] The two strands forming the double-stranded structure may be different parts of a larger RNA molecule, or they may be different RNA molecules. When 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 chain, the joined RNA strand is called 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. In some embodiments, a hairpin loop may contain 10 nucleotides or less. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 nucleotides.

[0101] 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 base pair count 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. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent includes a 5' overhang of at least one nucleotide. In specific embodiments, at least one strand includes a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent include an overhang of at least one nucleotide.

[0102] In one embodiment, the RNAi agent of the present invention is a dsRNA that interacts with a target RNA sequence, such as a SCAP target mRNA sequence, to induce cleavage of the target RNA, with each strand containing 19-23 nucleotides. Although theoretical constraints are not desired, the long double-stranded RNA introduced into the cell is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into a small interfering RNA of 19-23 base pairs with a characteristic 2-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA double strand, allowing the complementary antisense strand to induce 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).

[0103] 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 beyond 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.

[0104] 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.

[0105] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' and / or 5' ends, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at its 3' and / or 5' ends, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0106] In certain embodiments, the overhang of the sense strand, the antisense strand, or both may include an extended length greater than 10 nucleotides, for example, 1–30 nucleotides, 2–30 nucleotides, 10–30 nucleotides, or 10–15 nucleotides. In certain embodiments, the extended overhang is located on the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded sense strand. In certain embodiments, the extended overhang is located on the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 3' end of the double-stranded antisense strand. In certain embodiments, the extended overhang is located at the 5' end of the double-stranded antisense strand. In certain embodiments, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates. In certain embodiments, the overhang includes a self-complementary portion, and therefore the overhang has the ability to form a stable hairpin structure under physiological conditions.

[0107] The terms “blunt-terminated” or “blunt-terminated,” when used herein in reference to dsRNA, mean that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of a dsRNA may be blunt-terminated. If both ends of a dsRNA are blunt-terminated, it is said to be blunt-terminated. For clarification, a “blunt-terminated” dsRNA is a dsRNA where both ends are blunt-terminated, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded along their entire length.

[0108] The terms "antisense strand" or "guide strand" refer to an iRNA strand, such as a dsRNA, that contains a region substantially complementary to the target sequence, such as SCAP mRNA.

[0109] As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a target sequence, such as, for example, a SCAP 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 acceptable mismatches are in the terminal regions of the iRNA, e.g., within the range of 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0110] 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.

[0111] In the use of this specification, the term “cleavage region” refers to a region located directly adjacent to a cleavage site. A cleavage site is the target site where cleavage occurs. In some embodiments, the cleavage region includes one end of the cleavage site and three bases directly adjacent to the cleavage site. In some embodiments, the cleavage region includes one end of the cleavage site and two bases directly adjacent to the cleavage site. In some embodiments, the cleavage region is located, in particular, at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage region includes nucleotides 11, 12, and 13.

[0112] 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 be applied. 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.

[0113] 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, but generally five, four, three, or two or fewer, mismatched base pairs during double-stranded hybridization of up to 30 base pairs, while retaining the ability to hybridize 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 a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, is still referred to as “perfectly complementary” for the purposes described herein.

[0114] "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.

[0115] In this specification, the terms “complementary,” “fully complementary,” and “substantially complementary” may be used in relation to base matching between the sense strand and antisense strand 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.

[0116] As used herein, a polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide substantially complementary to a continuous portion of the mRNA in question (e.g., the mRNA encoding the SCAP gene). For example, a polynucleotide is complementary to at least a portion of SCAP mRNA if its sequence is substantially complementary to an undisrupted portion of the mRNA encoding the SCAP gene.

[0117] Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target SCAP sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target SCAP sequence and include a sequence of nucleotides that is at least about 80% complementary over equal regions of the nucleotide sequences of SEQ ID NOs. 1 to 13, or over their 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.

[0118] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target SCAP sequence and include one of the sense strand nucleotide sequences in any one of Table 2, Table 3, Table 5, or Table 6, or a fragment of one of the sense strand nucleotide sequences in any one of Table 2, Table 3, Table 5, or Table 6, and a continuous nucleotide sequence that is at least about 80% complementary over 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.

[0119] In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, wherein the antisense polynucleotide is identical to the target SCAP sequence, where the sense strand polynucleotide comprises a continuous nucleotide sequence that is at least about 80% complementary over an equal region of any one of the nucleotide sequences of SEQ ID NOs. 14-26, or over 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.

[0120] In one embodiment, at least partial suppression of SCAP gene expression is assessed by the decrease in the amount of SCAP mRNA that can be isolated or detected in a first cell or cell population where the SCAP gene is transcribed and which has been treated to inhibit SCAP gene expression, compared to a second cell or cell population (control cell) that is substantially identical to the first cell or cell population but has not undergone such treatment. The degree of inhibition is,

number

[0121] The phrase "bringing an RNAi agent, such as dsRNA, into contact with cells," as used herein, includes contacting cells by any possible means. Bringing an RNAi agent into contact with cells includes bringing iRNA into contact with cells in a test tube, or bringing iRNA into contact with cells in vivo. Contact may be direct or indirect. Therefore, for example, an RNAi agent may be placed in physical contact with cells by individually carrying out this method, or an RNAi agent may be placed in a situation that allows or can induce subsequent contact with cells.

[0122] Cell contact in vitro can be achieved, for example, by incubating cells with an RNAi agent. Cell contact in vivo can be achieved, for example, by injecting an RNAi agent into or near the tissue in which the cells are located, or by injecting an RNAi agent into another area, such as the bloodstream or subcutaneous space, to subsequently deliver the agent to the tissue in which the cells to be contacted are located. For example, an RNAi agent may contain and / or be coupled with a ligand that directs the RNAi agent to a target site, such as the liver, such as GalNAc3. Combinations of in vitro and in vivo contact methods are also possible. For example, cells may also be contacted with an RNAi agent in vitro and subsequently transplanted into a subject.

[0123] In one embodiment, contacting cells with iRNA includes “introducing” or “delivering iRNA to cells” by promoting or causing uptake or absorption into the cells. Absorption or uptake of iRNA may occur by natural diffusion or active cellular processes, or by auxiliaries or devices. Introduction of iRNA into cells may be in vitro and / or in vivo. For example, in vivo introduction, iRNA may be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further methods are described below herein and / or are known in the art.

[0124] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, iRNA or a plasmid from which iRNA is transcribed. LNPs are described, for example, in U.S. Patents No. 6,858,225, No. 6,815,432, No. 8,158,601, and No. 8,058,069 (all of which are incorporated herein by reference).

[0125] In the use of this specification, “Subject” means animals such as primates (humans, non-human primates such as monkeys and chimpanzees, etc.), mammals including non-primates (cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales, etc.), or birds (e.g., ducks or geese). In one embodiment, the subject is a human being treated or evaluated for a disease, disorder or condition that would benefit from reduced SCAP expression; a human being at risk of a disease, disorder or condition that would benefit from reduced SCAP expression; a human being having a disease, disorder or condition that would benefit from reduced SCAP expression; and / or a human being treated for a disease, disorder or condition that would benefit from reduced SCAP expression as described herein.

[0126] As used herein, the terms “to treat” or “to cure” refer to beneficial or desired outcomes, including, but not limited to, the reduction or improvement of one or more conditions associated with SCAP gene expression and / or SCAP protein production, such as fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). “Cure” may also mean an extension of survival compared to the predicted survival without treatment.

[0127] The term “reduce” in relation to the level of SCAP or disease marker or symptom in a subject means a statistically significant reduction in such level. The reduction 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 greater. In certain embodiments, the reduction is at least 20%. “Reduce” in relation to the SCAP level in a subject preferably means a reduction to a level that is considered to be within the normal range for an individual without such disorder.

[0128] In the context of this specification, “prevention” or “prevention” as used in relation to a disease, disorder, or condition that would benefit from reduced expression of the SCAP gene and / or production of the SCAP protein means a reduced likelihood of the subject developing symptoms associated with a disease, disorder, or condition, such as the presence of symptoms of SCAP gene expression, e.g., fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, liver inflammation, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). Effective prevention is considered to be the absence of the disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically recognized scale), or a delay in the onset of delayed symptoms (e.g., by several days, weeks, months, or years).

[0129] As used herein, the term “SCAP-related disease” refers to a disease or disorder caused by or associated with SCAP gene expression or SCAP protein production. The term “SCAP-related disease” includes diseases, disorders, or conditions that would benefit from reduced SCAP gene expression, replication, or protein activity. Non-exclusive examples of SCAP-related diseases include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatitis, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, atherosclerosis, pancreatitis, non-insulin-dependent diabetes mellitus, coronary heart disease, and cerebrovascular disease. In one embodiment, the SCAP-related disease is non-alcoholic fatty liver disease (NAFLD). In another embodiment, the SCAP-related disease is non-alcoholic steatohepatitis (NASH). In another embodiment, the SCAP-related disease is cirrhosis. In another embodiment, the SCAP-related disease is insulin resistance. In yet another embodiment, the SCAP-related disease is not insulin resistance. In one embodiment, the SCAP-related disease is obesity.

[0130] In one embodiment, the SCAP-associated disease is non-alcoholic fatty liver disease (NAFLD). As used herein, “non-alcoholic fatty liver disease” is interchangeable with the term “NAFLD” and refers to a disease defined by the presence of macrovascular steatosis in the presence of less than 20 gm of alcohol per day. NAFLD is the most common liver disease in the United States and is generally associated with insulin resistance / type 2 diabetes and obesity. NAFLD manifests as steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For more information on NAFLD, see Tolman and Dalpiaz (2007) Ther. Clin. Risk. Manag., 3(6):1153–1163 (the entire content of which is incorporated herein by reference).

[0131] As used herein, the terms “steatosis,” “hepatic steatosis,” and “fatty liver disease” refer to the accumulation of triglycerides and other fats within liver cells.

[0132] As used herein, the term “non-alcoholic steatohepatitis” or “NASH” refers to hepatitis and liver damage caused by the accumulation of fat in the liver. NASH is part of a group of conditions called non-alcoholic fatty liver disease (NAFLD). NASH is similar to alcoholic liver disease, but it can occur in people who drink little to no alcohol. The main feature of NASH is fat in the liver, accompanied by inflammation and damage. The vast majority of people with NASH feel fine and are unaware that they have a liver problem. Nevertheless, NASH can become serious and can lead to cirrhosis, in which the liver suffers permanent damage and scarring, and can no longer function normally. NASH is usually first suspected in people who have elevated liver test values, such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST), which are included in a routine blood test panel. NASH is suspected when further evaluation does not show a clear reason for liver disease (such as drug therapy, viral hepatitis, or excessive alcohol consumption), and when X-ray or imaging of the liver shows fat. The only way to confirm a diagnosis of NASH and distinguish it from simple fatty liver disease is through liver biopsy.

[0133] As used herein, the histologically defined term “cirrhosis” refers to a diffuse course of the liver characterized by fibrosis and changes from normal liver structure to structurally abnormal nodules.

[0134] As used herein, the term “serum lipids” refers to any significant lipids present in the blood. Serum lipids may be present in the blood either in free form or as part of protein complexes, such as lipoprotein complexes. Non-limiting examples of serum lipids include triglycerides (TGs), cholesterol, such as total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), and intermediate-density lipoprotein cholesterol (IDL-C).

[0135] When used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to provide treatment for the disease (e.g., by reducing, improving, or maintaining the symptoms of the existing disease or one or more symptoms of the disease) when administered to a subject with SCAP-associated disorder. “Therapeutic dose” may vary depending on the RNAi agent, how the drug is administered, the disease and its severity, as well as the subject’s medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and other treatments.

[0136] When used herein, "prophylactic effective dose" is intended to contain an amount of iRNA sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject with SCAP-related disorder. Disease improvement includes a slowing of the disease course or a reduction in the severity of the disease that develops later. The "prophylactic effective dose" may vary depending on the iRNA, the method of drug administration, the degree of disease risk, and the patient's medical history, age, weight, family history, genetic predisposition, type of prior or concomitant therapy, and other individual characteristics, if any, that are present.

[0137] The “therapeutic effective dose” or “preventive effective dose” may also refer to the amount of RNAi agent that produces several desired local or systemic effects with a reasonable benefit / risk ratio applicable to any therapeutic agent. The iRNA used in the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such therapies.

[0138] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with the tissues of human and animal subjects, provided that there is no excessive toxicity, irritation, allergic response, or other problems or complications that balance out in a reasonable benefit-to-risk ratio.

[0139] In the use of this specification, the term “pharmacoagulably acceptable carrier” means a pharmacoagulably acceptable material, composition, or vehicle, such as a liquid or solid extender, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or a solvent encapsulation material involved in transporting or delivering the compound of interest from one organ or body part to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the subject being treated. Some examples of materials that can serve as pharmacologically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium sulfate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) Polyols such as ricerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Water free of pyrogenic substances; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solutions; (21) Polyesters, polycarbonates and / or polyacid anhydrides; (22) Bulking agents such as polypeptides and amino acids; (23) Serum components such as serum albumin, HDL, and LDL; and (22) Other non-toxic and suitable substances used in pharmaceutical formulations.

[0140] As used herein, the term “sample” includes similar collections of bodily fluids, cells, or tissues isolated from a subject, as well as bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum and serosal fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples include samples from tissue, organs, or localized areas. For example, a sample may originate from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may be obtained from the liver (e.g., the whole liver or a specific segment of the liver or a specific type of cell within the liver, e.g., hepatocytes). In some embodiments, “sample obtained from subject” refers to blood or plasma collected from the subject. In further embodiments, “sample obtained from subject” refers to liver tissue (or a component thereof) or retinal tissue (or a component thereof) obtained from the subject.

[0141] II. The iRNA of the present invention Described herein are iRNAs that inhibit the expression of the SCAP gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits the expression of the SCAP gene in cells, such as cells in a subject, such as a mammal, including, for example, a human with a SCAP-related disorder such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during SCAP gene expression, the complementary region being about 30 nucleotides or less in length (for example, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less). Upon contact with cells expressing the SCAP gene, the iRNA inhibits the expression of the SCAP gene (e.g., human, primate, non-primate, or avian SCAP gene) by at least approximately 10%, as assayed by methods such as PCR or branched DNA (bDNA) based, or by protein-based methods such as immunofluorescence analysis using Western blotting or flow cytometry.

[0142] 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 SCAP 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.

[0143] 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 are 15-30 base pair lengths, 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 pair lengths. Intermediate ranges and lengths between the listed ranges and lengths are also intended to be part of the present invention.

[0144] 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.

[0145] In some embodiments, the dsRNA is about 15–23 nucleotides long, or about 25–30 nucleotides long. Generally, dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, 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 that is 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 target that is long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0146] 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- 33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15- 19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 2 It will also be recognized that double-stranded regions of approximately 9 to 36 base pairs, such as 0-28, 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 15-30 base pairs that targets a desired RNA for cleavage, for example, 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 SCAP gene expression are not generated in the target cell by cleaving larger dsRNAs.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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 sequence may be selected from the group of sequences provided in any one of Tables 2, 3, 5, and 6, and the corresponding nucleotide sequence of the antisense strand of the sense strand may be selected from the group of sequences in any one of Tables 2, 3, 5, and 6. In this embodiment, one of the two sequences is complementary to the other, and one of the sequences is substantially complementary to the mRNA sequence that occurs during SCAP gene expression. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in any one of Tables 2, 3, 5, and 6, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2, 3, 5, and 6. In one embodiment, substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, a substantially complementary sequence of the dsRNA is contained on a single oligonucleotide.

[0151] The sequences in Tables 2, 3, 5, and 6 are described as modified sequences and / or complexed sequences, but 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 unmodified, uncomplexed, and / or modified and / or complexed sequences shown in any one of Tables 2, 3, 5, and 6.

[0152] 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., (2001) EMBO J., 20:6877-6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures may 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 herein, the dsRNAs described herein may comprise at least one strand of a minimum length of 21 nucleotides. It can be reasonably expected that shorter double-stranded structures with only a few nucleotides missing from one or both ends may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein, whose ability to inhibit SCAP gene expression differs from dsRNAs containing the full-length sequence by only about 5, 10, 15, 20, 25, or 30% or less, are intended to be within the scope of the present invention.

[0153] Furthermore, the RNAs described herein identify sites in the SCAP transcript that are highly sensitive to RISC-mediated cleavage. Therefore, the present invention further features iRNAs that target these sites. In the use herein, an iRNA is said to target a specific site within the RNA transcript if it promotes cleavage of the transcript somewhere within a particular site. Such iRNAs generally consist of approximately 15 consecutive nucleotides from one of the sequences provided herein, ligated to an additional nucleotide sequence from a region adjacent to a select sequence in the SCAP gene.

[0154] 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 (e.g., by computer simulation) placing a given size “window” or “mask” (21 nucleotides as an 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, along with the identification of the best-performing sequences by systematic synthesis and testing (using assays described herein or known in the art) of the identified sequences, allows for the identification of RNA sequences that best mediate the inhibition of target gene expression when targeted with an iRNA agent. Therefore, while the sequences identified herein correspond to effective target sequences, it is intended that by progressively "shifting the window" by one nucleotide upstream or downstream of a given sequence, sequences with equivalent or better inhibitory properties can be identified, thereby achieving further optimization of inhibitory efficiency.

[0155] Furthermore, it is considered that further optimization of any sequence identified herein 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 inhibitory efficiency. Moreover, such optimized sequences may be modulated by further optimizing the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting specific sites or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) by, for example, introducing modified nucleotides described herein or known in the art, adding or modifying overhangs, or other modifications known in the art and / or considered herein.

[0156] The iRNA agents described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNA described herein contains 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 agent strand complementary to the SCAP gene region, the RNA strand 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 SCAP gene expression. Examining the effectiveness of mismatched iRNAs in inhibiting SCAP gene expression is particularly important when specific complementary regions of the SCAP gene are known to have polymorphic sequence variations within the population.

[0157] 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. The iRNA of the present invention that is "substantially all of its nucleotides modified" is mostly modified but not completely modified and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0158] The nucleic acids discussed herein may 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. For example, modifications include terminal modifications such as 5'-end modifications (phosphorylation, conjugation, inversion) or 3'-end modifications (conjugation, DNA nucleotides, inversion, etc.); base modifications such as substitution, base removal (debasing nucleotide), or conjugated bases, for example, at stabilizing bases, destabilizing bases, or bases that form base pairs with the repertoire of expanding partners; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and main chain modifications, including modifications or substitutions 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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 are directly or indirectly bound 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 the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0164] 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.

[0165] 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-C 10 Alkyl, or C2-C 10 It can be an alkenyl or alkinyl. An exemplary suitable modification is O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n One example is CH3)2 (wherein n and m are 1 to approximately 10). In another embodiment, the dsRNA contains one of the following at the 2' position: C1~C 10Lower alkyl groups, substituted lower alkyl groups, alkaryl groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, interveners, groups that improve the pharmacokinetic properties of iRNA, or groups that improve the pharmacodynamic properties of iRNA, and other substituents having similar properties. In some embodiments, the modification includes 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 include the O(CH2)2ON(CH3)2 group, as described in the following examples herein, which is 2'-dimethylaminooxyethoxy, 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. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families); 2'-alkoxyalkyls; and 2'-NMA (N-methylacetamide).

[0166] 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 RNA, 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.

[0167] The iRNAs of the present invention may also include modifications or substitutions of nucleic acid bases (often simply referred to as "bases" in the art). In the use herein, "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 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (psoidouracil); 4-thio Examples include ouracil; 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 other synthetic and natural nucleic acid bases such as 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., (1991) Angewandte Chemie, International Edition, 30:613; and those disclosed by Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in 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.

[0168] 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. Examples of U.S. patent specifications include, but are not limited to, U.S. Patent Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088.

[0169] The iRNA of the present invention can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, where the ribose moiety contains an additional crosslink connecting the 2' and 4' carbons. This structure effectively "locks" the ribose into a 3' terminal configuration. Adding 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).

[0170] The iRNA of the present invention 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 molecule containing a bridge that connects two carbon atoms of a sugar ring, thereby forming a bicyclic system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present invention include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety with a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose within the 3'-end (endo) conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce nonspecific 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, but are not limited to, nucleosides containing bridges between 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides containing 4'-2' bridges.Examples of such 4'-2' bridged bicyclic nucleosides include 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. 8,278,28 See Specification No. 3); 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' (where 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). The entire contents of each of the above-mentioned items are incorporated herein by reference.

[0171] Additional representative U.S. patents and publications teaching the preparation of locked nucleic acid nucleotides are, as incorporated herein by reference in their entirety, U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; and 7,084,125; Examples include, but are not limited to, U.S. Patent Application Publication No. 7,399,845; No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281.

[0172] For example, any of the aforementioned bicyclic nucleosides having one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see International Publication No. 99 / 14226).

[0173] The iRNA of the present invention 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 comprising 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”.

[0174] The iRNA of the present invention may also include one or more “contour-fixed nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable configuration, increasing its hybridization affinity with mRNA. The linker is long enough to place the oxygen in an optimal position in terms of stability and affinity, thus reducing ribose ring puckering.

[0175] Representative documents that provide instruction on 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).

[0176] In some embodiments, the iRNA of the present invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which one of the sugar bonds is removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) is removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, as incorporated herein by reference).

[0177] Representative documents teaching some of the aforementioned preparations of CRN include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and International Publication No. 2013 / 036868, which are incorporated herein by reference in their entirety.

[0178] 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). Disclosure of these modifications can be found in International Publication No. 2011 / 005861.

[0179] Other modifications of the iRNA of the present invention include 5'-phosphates or 5'-phosphate mimes, for example, 5'-terminal phosphates or phosphate mimes on the antisense strand of an RNAi agent. Suitable phosphate mimes are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, which is incorporated herein by reference in its entirety.

[0180] A. Modified iRNA containing the motif of the present invention In certain embodiments of the present invention, the double-stranded RNAi agents of the present invention include chemically modified agents, for example, as disclosed in International Publication No. 2013 / 075035, filed on November 16, 2012, which is incorporated herein by reference in its entirety. Excellent results can be achieved by introducing one or more motifs of three identical modifications to three consecutive nucleotides on the sense and / or antisense strands of the RNAi agent, particularly at or near the cleavage site, as shown herein and in International Publication No. 2013 / 075035. In addition, in some embodiments, the sense and antisense strands of the RNAi agent may be completely modified. The introduction of these motifs disrupts the modification pattern of the sense and / or antisense strands, if present. The RNAi agent may be optionally conjugated, for example, with a GalNAc derivative ligand on the sense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0181] More specifically, surprisingly, it was found that when the sense and antisense strands of a double-stranded RNAi agent are completely modified to have one or more motifs of three identical modifications to a cleavage site on at least one strand of the RNAi agent or to three consecutive nucleotides near that site, the gene silencing activity of the RNAi agent is greatly increased.

[0182] Accordingly, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the SCAP gene) in vivo. The 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.

[0183] The sense and antisense strands typically form a duplex 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.

[0184] 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 the same length being misaligned. The overhang may form a mismatch with the target mRNA, or it may be complementary to or different from the target gene sequence. The first and second strands may be linked, for example, by additional bases forming a hairpin, or by other non-base linkers.

[0185] In one embodiment, the nucleotides in the overhang region of the RNAi agent may be independently modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications such as 2-F, 2'-O methyl, thymidine (T), and any combination thereof. For example, TT may be an overhang sequence to any 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.

[0186] The 5' or 3' end of the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region comprises two nucleotides having a phosphorothioate between them, and 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.

[0187] RNAi agents contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, the single-stranded overhang may be located at the 3' end of the sense strand, or also at the 3' end of the antisense strand. RNAi may also be located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa, with a blunt end. 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 impose theoretical constraints, the asymmetric blunt ends of the 5' end and 3' end overhangs of the antisense strand are favorable for introducing guide strands into RISC processes.

[0188] In one embodiment, the RNAi agent is a 19-nucleotide long double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications to three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications to three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0189] In another embodiment, the RNAi agent is a 20-nucleotide blunt-ended double strand, the sense strand containing at least one motif of three 2'-F modifications to three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications to three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0190] In another embodiment, the RNAi agent is a 21-nucleotide blunt-ended double strand, the sense strand containing at least one motif of three 2'-F modifications to three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications to three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0191] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications to three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications to three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with one end of the RNAi agent being blunt and the other end comprising a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is at the 3' end of the antisense strand. If the 2-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide interbonds between the three terminal nucleotides, where two of the three nucleotides are the overhang nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. 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 in the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in alternating motifs. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0192] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotides long and starting from the 5' terminal nucleotide (position 1), with positions 1-23 of the first strand containing at least 8 ribonucleotides; the antisense strand being 36-66 nucleotides long and starting from the 3' terminal nucleotide, with at least 8 ribonucleotides at positions paired with positions 1-23 of the sense strand to form a double helix; at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand, and six or fewer consecutive 3' terminal nucleotides are unpaired with the sense strand, thus forming a 1-6 nucleotide 3' single-stranded overhang; where the 5' end of the antisense strand is unpaired with the sense strand, forming a 10-30 consecutive nucleotide overhang; Because it contains ocides, it forms a single-stranded 5' overhang of 10-30 nucleotides; when the sense and antisense strands are aligned for maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand base-pair with the nucleotides of the antisense strand, thus forming a substantially doubled region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so that the doubled nucleic acid reduces the expression of the target gene when introduced into mammalian cells; the sense strand contains at least one motif of three 2'-F modifications to 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 to three consecutive nucleotides at or near the cleavage site.

[0193] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 and a maximum of 29 nucleotides, and a second strand having a length of up to 30 nucleotides, comprising a motif of at least one of three 2'-O-methyl modifications to three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, and the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the second strand length so that when the RNAi agent is introduced into mammalian cells, the expression of the target gene is reduced, here, dicer cleavage of the RNAi agent preferentially yields the siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, the RNAi agent further comprises a ligand.

[0194] In one embodiment, the sense strand of the RNAi agent comprises at least one motif of three identical modifications to three consecutive nucleotides, one of which is located at a cleavage site of the sense strand.

[0195] In one embodiment, the antisense strand of the RNAi agent comprises at least one motif of three identical modifications to three consecutive nucleotides, one of which is located at or near the cleavage site of the antisense strand.

[0196] For RNAi agents with a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically near 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, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0197] The sense strand of an RNAi agent may contain at least one motif of three identical modifications to three consecutive nucleotides at the cleavage site of the strand; the antisense strand may have at least one motif of three identical modifications to three consecutive nucleotides at or near the cleavage site of the strand. 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 in the sense strand and one motif of three nucleotides in the antisense strand have at least one nucleotide duplication, 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 may be duplicated, or all three nucleotides may be duplicated.

[0198] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications to three consecutive nucleotides. The first motif may be located at or near the cleavage site of the strand, and the other motifs may be wing modifications. In this specification, the term “wing modification” refers to a motif located in a different part of the strand, away from the motif at or near the cleavage site of the same strand. Wing modifications are adjacent to the first motif or separated by at least one nucleotide. If the motifs are directly adjacent to each other, their chemical structures are different; if the motifs are separated by one or more nucleotides, their chemical structures may be the same or different. Two or more wing modifications may be present. For example, if two wing modifications are present, each wing modification may be located at one end relative to the first motif at or near the cleavage site, or on either side of the lead motif.

[0199] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications to three consecutive nucleotides, with at least one of the motifs located at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in a sequence similar to the wing modifications that may be present on the sense strand.

[0200] In one embodiment, the wing modification to the sense or antisense strand of the RNAi agent typically does not include the first one or two ends at the 3' end, 5' end, or both ends of the strand.

[0201] In another embodiment, the wing modification to the sense or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0202] If the sense strand and antisense strand of the RNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0203] If the sense strand and antisense strand of an RNAi agent each contain at least two wing modifications, the sense strand and antisense strand can be aligned such that two modifications from one strand are each located at one end of the double-stranded region, having one, two, or three nucleotide duplicates; two modifications from one strand are each located at the other end of the double-stranded region, having one, two, or three nucleotide duplicates; and two modifications from one strand are located on each side of the read motif, having one, two, or three nucleotide duplicates in the double-stranded region.

[0204] In one embodiment, all 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 with the same or different modifications, which may include one or more alterations of one or both of the unbound phosphate oxygen and / or one or more bound phosphate oxygens; alterations of components of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar; large-scale substitution of the phosphate moiety by a "dephospho" linker; modification or substitution of native bases; and substitution or modification of the ribose-phosphate backbone.

[0205] Since nucleic acids are polymers of subunits, many modifications, such as modifications to bases, phosphate moieties, or unbound oxygen atoms of phosphate moieties, are located at repeating positions within the nucleic acid. In some cases, modifications may be present at all desired positions in the nucleic acid, but often this is not 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 the double-stranded region, the single-stranded region, or both. Modifications may be present only in the double-stranded region of RNA, or only in the single-stranded region of RNA. For example, phosphorothioate modifications at unbound oxygen positions may be present at only 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 the double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends can be phosphorylated.

[0206] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes (surrogates) in single-stranded overhangs, e.g., 5' or 3' overhangs, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or part of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of ribose sugars by modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modifications instead of ribosaccharides in nucleic acid bases, and modifications of phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0207] In one embodiment, each residue in the sense and antisense chains is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. 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.

[0208] At least two distinct modifications are typically present in the sense and antisense chains. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, or other modifications.

[0209] In one embodiment, N a and / or N b This includes alternating pattern modifications. In the usage herein, the term “alternating motif” refers to a motif having one or more modifications, where each modification is present in alternating nucleotides on a single chain. Alternating nucleotides may be one every other nucleotide, one 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···”.

[0210] The types of modifications included in the alternating motifs may be the same or different. For example, if A, B, C, and D each represent one type of modification to their respective nucleotides, the alternating pattern, i.e., the modifications every other nucleotide, may be the same, but each of the sense strand or antisense strand may be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0211] In one embodiment, the RNAi agent of the present invention includes an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. This shift may be such that the nucleotide modification groups on the sense strand correspond to different modification groups on the nucleotides of the antisense strand, or vice versa. For example, when the sense strand pairs with the antisense strand in a dsRNA double strand, the alternating motif on the sense strand may begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif on the antisense strand may begin with "BABABA" from 5' to 3' of the strand within the double-stranded region. As another example, the alternating motif on the sense strand may begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif on the antisense strand may begin with "BBAABBAA" from 5' to 3' of the strand within the double-stranded region, thereby resulting in a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

[0212] In one embodiment, the RNAi agent comprises 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 base-pairs 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.

[0213] The introduction of one or more motifs of three identical modifications to three consecutive nucleotides into the sense strand and / or antisense strand disrupts the initial modification pattern present in the sense strand and / or antisense strand. Surprisingly, this disruption of the modification pattern in the sense strand and / or antisense strand by introducing one or more motifs of three identical modifications to three consecutive nucleotides into the sense strand and / or antisense strand enhances gene silencing activity against the target gene.

[0214] In one embodiment, when three identical modification motifs for three consecutive nucleotides are introduced into any of the chains, the modifications of nucleotides adjacent to the motif are different from the modifications of the motif. For example, a portion of the sequence containing the motif is "···N a YYYN b ..." where "Y" represents the modification of three identical modification motifs to three consecutive nucleotides, and "N a " and "N b " represents a modification of a nucleotide adjacent to the motif "YYY", which is different from the modification of Y, and N a and N b These may be the same or different modifications. Or, N a and / or N b This may or may not exist if a wing modifier is present.

[0215] The RNAi agent may further contain at least one phosphorothioate or methylphosphonate internucleotide bond. The modification of the phosphorothioate or methylphosphonate internucleotide bond may be present on any nucleotide in the sense strand, the antisense strand, or both strands, at any position in the strand. For example, the modification of the internucleotide bond may be present on all nucleotides in the sense strand and / or antisense strand; each modification of the internucleotide bond may be present in an alternating pattern in the sense strand and / or antisense strand; or the sense strand or antisense strand may contain modifications of both internucleotide bonds in an alternating pattern. The alternating pattern of modifications of the internucleotide bonds on the sense strand may be the same as or different from that on the antisense strand, and the alternating pattern of modifications of the internucleotide bonds on the sense strand may have a shift relative to the alternating pattern of modifications of the internucleotide bonds on the antisense strand. In one embodiment, the double-stranded RNAi agent contains 6-8 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.

[0216] In one embodiment, the RNAi includes modifications of phosphorothioate or methylphosphonate internucleotide bonds in the overhang region. For example, the overhang region may include two nucleotides having a phosphorothioate or methylphosphonate internucleotide bond between them. Furthermore, modifications of internucleotide bonds can be carried out to link the overhang nucleotides to the terminal paired nucleotides in the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, additional phosphorothioate or methylphosphonate internucleotide bonds may be present to link the overhang nucleotides to the paired nucleotides adjacent to the overhang nucleotides. For example, at least two phosphorothioate internucleotide bonds may be present between the three terminal nucleotides, where two of the three nucleotides 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.

[0217] 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, two of which are the overhang nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhang nucleotide. 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.

[0218] In one embodiment, the RNAi agent includes mismatches with the target, intra-double-strand mismatches, or combinations thereof. Mismatches may occur in overhang regions or double-strand regions. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., with respect to the free energy of binding or dissociation of a particular pair, the simplest method being to examine each pair individually, although similar or equivalent analyses may be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical or non-canonical pairings (as described elsewhere in this specification), are preferred over canonical (A:T, A:U, G:C) pairings; pairings containing universal bases are preferred over canonical pairings.

[0219] In one embodiment, the RNAi agent comprises at least one of the first one, two, three, four, or five base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the group A:U, G:U, and I:C, and a mismatch pair to facilitate the dissociation of the antisense strand at the 5' end of the double-stranded region, such as a non-canonical or non-canonical pair or a pair containing a universal base.

[0220] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0221] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In yet another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides for the 3' ends of the sense and / or antisense strands.

[0222] In one embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) It can be expressed by, in the formula, i and j are independently either 0 or 1; p and q are each independently between 0 and 6; each N a Each represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b Each represents an oligonucleotide sequence containing 0 to 10 modified nucleotides independently; each n p and n q These independently represent overhanging nucleotides; Here, Nb and Y do not have the same modifications; XXX, YYY, and ZZZ each independently represent one motif of three identical modifications to three consecutive nucleotides. Preferably, all YYY nucleotides are 2'-F modified nucleotides.

[0223] In one embodiment, N a and / or N b This includes alternating pattern modifications.

[0224] In one embodiment, the YYY motif is located at or near the sense strand cleavage site. For example, if 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, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end (for example, 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).

[0225] 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'n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3' (Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) It can be represented by [this].

[0226] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0227] If the sense chain is represented by formula (Ic), then N bThis represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0228] If the sense chain is represented by formula (Id), then each N b This independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0229] Each of X, Y, and Z may be the same as or different from one another.

[0230] In other embodiments, i is 0, j is 0, and the sense chain is given by the following equation: 5'n p -N a -YYY-N a -n q 3' (Ia) It can be represented by [this].

[0231] If the sense chain is represented by equation (Ia), then each N a This can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0232] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N a '-n p '3' (II) It can be expressed by, in the formula, k and l are independently either 0 or 1; p' and q' are each independently between 0 and 6; each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p 'and n q ' independently represents an overhanging nucleotide; Here, N b 'and Y' do not have the same modifier; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications to three consecutive nucleotides.

[0233] In one embodiment, N a 'and / or N b ' includes alternating pattern modifications.

[0234] The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, if the 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; or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end; or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end. Preferably, the Y'Y'Y' motif is located at positions 11, 12, 13.

[0235] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0236] 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.

[0237] Therefore, the antisense chain is given by the following formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb); 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc); or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (IId) It can be represented by [this].

[0238] If the antisense chain is expressed as equation (IIb), then N b ' represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0239] If the antisense chain is expressed as equation (IIc), then N b ' represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0240] If the antisense chain is expressed as equation (IId), then each N b' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, each N b It is 0, 1, 2, 3, 4, 5, or 6.

[0241] In other embodiments, k is 0, l is 0, and the antisense chain is given by the following formula: 5'n q’ -N a’ -Y'Y'Y'-N a’ -n p’ 3' (Ia) It can be represented by [this].

[0242] If the antisense chain is represented by equation (IIa), then each N a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0243] Each of X', Y', and Z' may be the same as or different from one another.

[0244] Each nucleotide in the sense and antisense strands may be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands may be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0245] In one embodiment, the sense strand of the RNAi agent may include YYY motifs located at positions 9, 10, and 11 of the strand, starting from the first nucleotide from the 5' end, or optionally, starting from the first paired nucleotide in the double-stranded region from the 5' end; Y represents a 2'-F modification. The sense strand may further include an XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region; XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0246] In one embodiment, the antisense strand may include Y'Y'Y' motifs located at positions 11, 12, and 13 of the strand, counting from the first nucleotide from the 5' end, or optionally, counting from the first paired nucleotide in the double-stranded region from the 5' end; Y' represents a 2'-O-methyl modification. The antisense strand may further include X'X'X' or Z'Z'Z' motifs as wing modifications at the opposite end of the double-stranded region; X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0247] Each sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0248] Therefore, the RNAi agent for use in the method of the present invention may include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double strand is given by formula (III): Sense: 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p -Na '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) It is expressed by, 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 N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two different modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ',n p , n q ', and n q Each of these may or may not exist, and independently represents an overhang nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications to three consecutive nucleotides.

[0249] 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.

[0250] An example combination of sense and antisense strands that form an RNAi double helix is ​​given by the following formula: 5'n p -N a -YYY-N a-n q 3' 3'n p’ -N a’ -Y'Y'Y'-N a’ n q’ 5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ n q’ 5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a -n q '5' (IIId) Includes.

[0251] If an RNAi agent is represented by formula (IIIa), then each N a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0252] If an RNAi agent is represented by formula (IIIb), then each N b Each N independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0253] When an RNAi agent is represented by formula (IIIc), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0254] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents oligonucleotide sequences containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ' independently represents oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b Each of these independently includes alternating pattern modifications.

[0255] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), each of X, Y, and Z may be the same as or different from one another.

[0256] If an RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), then at least one Y nucleotide may form a base pair with one of the Y' nucleotides; or at least two Y nucleotides may form base pairs with the corresponding Y' nucleotides; or all three Y nucleotides may form base pairs with the corresponding Y' nucleotides.

[0257] If an RNAi agent is represented by formula (IIIb) or (IIId), then at least one Z nucleotide may form a base pair with one of the Z' nucleotides; or at least two Z nucleotides may form base pairs with the corresponding Z' nucleotides; or all three Z nucleotides may form base pairs with the corresponding Z' nucleotides.

[0258] If an RNAi agent is represented by formula (IIIc) or (IIId), then at least one X nucleotide may form a base pair with one of the X' nucleotides; or at least two X nucleotides may form base pairs with the corresponding X' nucleotides; or all three X nucleotides may form base pairs with the corresponding X' nucleotides.

[0259] In one embodiment, modifications to the Y nucleotide differ from modifications to the Y' nucleotide, modifications to the Z nucleotide differ from modifications to the Z' nucleotide, and / or modifications to the X nucleotide differ from modifications to the X' nucleotide.

[0260] In one embodiment, if the RNAi agent is represented by formula (IIId), then N a The modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, if the RNAi agent is represented by formula (IIId), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p' is bound to an adjacent nucleotide via a phosphorothioate bond. In another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described later). In another embodiment, if the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The nucleotide is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0261] In one embodiment, if the RNAi agent is represented by formula (IIIa), then N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p The nucleotide is bound to an adjacent nucleotide via a phosphorothioate bond, and the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0262] In one embodiment, the RNAi agent is a multimer comprising at least two double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), which are linked by a linker. The linker may be cleavable or incleavable. Optionally, the multimer further comprises ligands. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0263] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more double strands represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double strands are linked by linkers. The linkers may be cleavable or incleavable. Optionally, the multimer further comprises ligands. Each double strand may target the same gene or two different genes; or each double strand may target the same gene at two different target sites.

[0264] In one embodiment, two RNAi agents, represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), are conjugated to each other at their 5' ends and to one or both of their 3' ends, and are optionally conjugated to a ligand. Each RNAi agent may target the same gene or two different genes; or each RNAi agent may target the same gene at two different target sites.

[0265] Various publications describe multimeric RNAi agents that can be used in the methods of the present invention. Such publications include International Publication No. 2007 / 091269, U.S. Patent No. 7858769, International Publication Nos. 2010 / 141511, 2007 / 117686, 2009 / 014887, and 2011 / 031520, the full contents of which are incorporated herein by reference.

[0266] As will be described in more detail below, RNAi agents containing the conjugation of one or more carbohydrate moieties to the RNAi agent can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety will be bound to a modified subunit of the RNAi agent. For example, one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier, which will be bound to the carbohydrate ligand. Ribonucleotide subunits in which the ribose sugar of the subunit is thus substituted are referred herein to as ribose-substituted modified subunits (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms or heterocyclic systems, i.e., one or more ring atoms may be heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic system or may contain two or more rings, e.g., a fusion ring. The cyclic carrier may be a fully saturated cyclic system or may contain one or more double bonds.

[0267] Ligands can be bound 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., a phosphate of ribonucleic acid, or a modified phosphate (e.g., sulfur-containing) skeleton. In some embodiments, “tethering linkage site” (TAP) refers to a constituent ring atom of the cyclic carrier that binds to a select moiety, e.g., a carbon atom or heteroatom (different from the atom that donates the skeletal linkage sites). The select moiety may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Optionally, the select moiety is bound to the cyclic carrier by an intervention tether. Therefore, cyclic supports often contain functional groups, such as amino groups, or generally provide suitable bonds for incorporating or anchoring another chemical entity, such as a ligand, to the constitutive ring.

[0268] The RNAi agent may be conjugated to the ligand via a carrier, which may be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinil, pyrazolidinil, imidazolinil, imidazolidinil, piperidinil, piperazinil, [1,3]dioxolane, oxazolidinil, isoxazolidinil, morpholinil, thiazolidinil, isothiazolidinil, quinoxalinil, pyridadinil, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0269] In certain embodiments, the RNAi agent for use in the method of the present invention is an agent selected from the group of agents listed in either Table 2 or 3. These agents may further contain ligands.

[0270] IV. Ligand-coupled iRNAs Another modification of the iRNA of the present invention involves chemically linking one or more ligands, moieties, or complexes to the RNA, thereby enhancing the activity, cell distribution, or intracellular uptake of the iRNA. Such parts include lipid portions such as the cholesterol portion (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86:6553-6556); cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NYAcad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770) and thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538); and dodecanediol or undecyl residues (Saison-Behmoaras et al. Aliphatic chains such as al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54); phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al. al., (1995) Nucleosides & Nucleotides, 14:969-973); or adamantane acetate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654); palmityl moiety (Mishra et al., (1995) Biochim. Biophys.Examples include, but are not limited to, octadecylamine or the hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0271] 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.

[0272] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, 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.

[0273] The ligand may also include a targeting group such as an antibody that binds to a specific cell type, such as kidney cells, or a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein. The targeting group may be thyroid-stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetylglucosamine, polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0274] 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, and O3-(oleoyl)lithocholic acid. Examples include O3-(oleoyl)cholenic 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.

[0275] Ligands can be proteins, such as glycoproteins; peptides, such as molecules having 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.

[0276] 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.

[0277] 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.

[0278] The ligand-conjugated oligonucleotide of the present invention may be synthesized by using an oligonucleotide having a pendant-reactive functional group, such as one derived from the addition of a binding molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with a commercially available ligand, a synthesized ligand having any of the various protecting groups, or a ligand having an attachable binding site.

[0279] The oligonucleotides used in the complex of the present invention may, conveniently and conventionally, be produced by well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including Applied Biosystems (Foster City, Calif.). In addition or alternatively, any other means known in the art for such synthesis may be used. Similar techniques are also known to be used to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0280] 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.

[0281] 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.

[0282] 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. For example, target tissues may include liver cells, including those of the liver. Other molecules capable of binding to HSA can also be used as ligands. For example, neproxine or aspirin may 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.

[0283] For example, lipid-based ligands can be used for inhibition, such as 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 kidney and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the complex to the kidney.

[0284] In a preferred embodiment, a 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.

[0285] 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 parts that target renal cells may be used instead of or in addition to the lipid-based ligand.

[0286] In another embodiment, ligands are portions of vitamins, for example, that are taken up by target cells such as proliferating cells. These are particularly useful in 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, and pyridoxal, or other vitamins or nutrients that are taken up by target cells such as liver cells. HSA and low-density lipoprotein (LDL) are also examples.

[0287] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, preferably a helical cell permeabilizer. Preferably, the cell permeabilizer is amphiphilic. Exemplary cell permeabilizers are peptides such as tat or antennopedia. If the cell permeabilizer is a peptide, it may be modified, including peptidyl mimetic, inverted isomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having lipophilic and oleophobic phases.

[0288] The ligand may be a peptide or a peptide mimetic. Peptidimides (also referred to herein as oligopeptide mimes) are molecules that can fold into a defined three-dimensional structure similar to natural peptides. The addition of peptides and peptide mimes to iRNA agents may affect the pharmacokinetic distribution of the iRNA, such as by enhancing cell recognition and absorption. The peptide or peptide mimetic moiety may be approximately 5 to 50 amino acid lengths, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths.

[0289] 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 transition sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 27). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 28)) 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: 29)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 30)) 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 through 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.

[0290] 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.

[0291] "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 bifidopphipathic 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).

[0292] 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, each 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 5 or more C molecules (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (e.g., C5, C6, C7, or C8).

[0293] In one embodiment, the carbohydrate complex used in the composition and method of the present invention is a monosaccharide. In another embodiment, the carbohydrate complex used in the composition and method of the present invention is [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0294] In one embodiment, the monosaccharide is [ka] These include N-acetylgalactosamine.

[0295] Other representative carbohydrate complexes used in the embodiments described herein include, but are not limited to, [ka] (When one of X or Y is an oligonucleotide, the other is hydrogen.)

[0296] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is bound to the iRNA agent of the present invention via a trivalent linker.

[0297] In one embodiment, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative conjugated to an iRNA agent. In another embodiment, the double-stranded RNAi agent of the present invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently conjugated to multiple nucleotides of the double-stranded RNAi agent by multiple monovalent linkers.

[0298] In some embodiments, for example, when the two strands of the iRNA agent of the present invention are part of a larger molecule that forms a hairpin loop containing multiple unpaired nucleotides, linked by an unbroken nucleotide chain between the 3' end of one strand and the 5' end of the other strand, each of the unpaired nucleotides within the hairpin loop may independently contain GalNAc or a GalNAc derivative linked via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the strands of the double helix.

[0299] In some embodiments, the carbohydrate complex further comprises one or more of the above-mentioned additional ligands, such as PK regulators and / or cell-permeable peptides.

[0300] Further carbohydrate complexes (linkers) suitable for use in the present invention are those described in International Publication Nos. 2014 / 179620 and 2014 / 179627, which are incorporated herein by reference in their entirety.

[0301] D. Linker In some embodiments, the complexes or ligands described herein may be attached to iRNA oligonucleotides by various linkers that may be cleavable or incleavable.

[0302] 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, aryl, heteroaryl, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroarylalkyls, alkylheteroarylalkenyls, alkylheteroarylalkynyls This includes, but is not limited to, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkylhetrocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylhetroaryl, alkenyl heteroaryl, alkynylhereroaryl, etc., and one or more methylene groups are O, S, S(O), SO2, N(R) 8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (wherein R8 The linker can be interrupted or terminated by hydrogen, acyl, aliphatic or substituted aliphatic atoms. In one embodiment, the linker is 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 17 atoms, or 8 to 16 atoms.

[0303] 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 (e.g., one that mimics or can be selected to mimic intracellular conditions) than in the target blood or under a second standard condition (e.g., one that mimics or can be selected to mimic conditions found in blood or serum).

[0304] 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.

[0305] 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.

[0306] 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 be linked to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and therefore the linker is 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.

[0307] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synovial cells.

[0308] 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, where the first condition is selected to demonstrate cleavage in target cells, and the second condition is selected to demonstrate cleavage in other tissues or in biological fluids such as blood or serum. Evaluations can be performed in cell-free systems, in cells, in cell cultures, in 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).

[0309] 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. A single 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.

[0310] ii. Phosphate-based cleavable linking groups In another embodiment, the cleavable linker includes 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-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0311] 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, 5.0 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 a cleavage environment for 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.

[0312] 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.

[0313] 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.

[0314] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA-carbohydrate complexes having linkers of the compositions and methods of the present invention include, but are not limited to, [ka] [ka] (When one of X or Y is an oligonucleotide, the other is hydrogen.)

[0315] 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.

[0316] In one embodiment, the dsRNA of the present invention is of formula (XXXII) to (XXXV), [ka] (In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent each occurrence from 0 to 20, and the repeating units may be identical or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C Each occurrence is independently of the others: absence, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each occurrence is independently of the others: absent, alkylene, substituted alkylene, and one or more methylene groups: O, S, S(O), SO2, N(R) N ), C(R')=C(R''), C≡C or C(O) may interrupt or terminate; R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each of these is independent of the others, either not existing or existing as NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a)C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocycline; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, each occurrence is independently a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a It is conjugated with a divalent or trivalent branched linker, selected from a group of structures represented by either H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are represented by formula (XXXVI), [ka] (In the formula, L 5A , L 5B and L 5C It is particularly useful when used in conjunction with RNAi agents to inhibit the expression of target genes (such as monosaccharides represented by GalNAc derivatives).

[0317] 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.

[0318] 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.

[0319] 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.

[0320] In the context of this invention, a "chimeric" iRNA compound or "chimera" 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, shorter iRNAs often yield comparable results compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of RNA targets can conventionally be detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0321] 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 implementing such conjugation 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 (Res.,1992,20:533), for example, 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. It contains (al., Nucleosides & 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 groups are then reacted with the molecule to be conjugated 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.

[0322] IV. Delivery of the iRNA of the Invention The delivery of the iRNA of the present invention to cells within a subject, such as a human subject (e.g., a subject with SCAP-related disorders, such as non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH), or any 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.

[0323] 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, W. J. 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; either method acts 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, iRNAs that counteract ApoB, which is conjugated to the lipophilic cholesterol portion, were systemically injected into mice, resulting in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of iRNAs 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 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, for example, 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. 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, DA. 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 their entirety in U.S. Patent No. 7,427,605, which is incorporated herein by reference.

[0324] A. Vector encoding the iRNA of the present invention SCAP gene-targeting iRNAs can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International 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 (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

[0325] 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.

[0326] 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 be 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.

[0327] 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 considerations regarding vectors and constructs are known in the art.

[0328] V. Pharmaceutical Composition of the Present Invention The present invention also includes pharmaceutical compositions and formulations containing the iRNA of the present invention. In one embodiment, the present invention provides a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for the treatment of diseases or disorders related to the expression or activity of the SCAP gene, such as SCAP-related diseases, such as non-alcoholic fatty liver disease (NAFLD), such as non-alcoholic steatohepatitis (NASH).

[0329] Such pharmaceutical compositions are formulated based on the method of delivery. One example is a composition formulated for systemic administration via parenteral delivery, for example, by intravenous (IV), intramuscular (IM), or subcutaneous (subQ) delivery. Another example is a composition formulated for direct delivery to the liver by infusion, such as by continuous pump infusion.

[0330] The pharmaceutical composition of the present invention can be administered in a dose sufficient to inhibit the expression of the SCAP gene. Generally, a preferred dose 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. Typically, a preferred dose of the iRNA of the present invention may range from about 0.1 mg / kg to about 5.0 mg / kg, preferably about 0.3 mg / kg and about 3.0 mg / kg.

[0331] Repeated-dose regimens may include periodic, for example, therapeutic doses of iRNA every other day to every year. In certain embodiments, iRNA is administered approximately every month to approximately every three months (i.e., approximately once every three months).

[0332] After the initial treatment regimen, the medication can be administered at a lower frequency.

[0333] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more subdoses spaced appropriately throughout the day, or even by continuous infusion or delivery from a controlled-release formulation. In this case, the iRNA contained in each subdose must correspondingly be less than the total daily dose. The dosage unit can also be compounded for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites, such as those that may be used with the drugs of the present invention. In this embodiment, the dosage unit contains a number of corresponding daily doses.

[0334] In other embodiments, since a single dose of the pharmaceutical composition can have a long duration of effect, subsequent doses are administered at intervals of 3, 4, or 5 days or less, or at intervals of 1, 2, 3, or 4 weeks or less. In some embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once a week. In other embodiments of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once every two months.

[0335] Those skilled in the art will understand, but will not limit, that certain factors, including 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 a subject with a therapeutically effective dose of the composition may consist of a single treatment or a series of treatments. Estimations of effective doses and in vivo half-lives for individual iRNAs incorporated in this invention can be made using conventional methods or based on in vivo studies using appropriate animal models, as described in other parts of this specification.

[0336] Advances in mouse genetics have led to the creation of numerous mouse models for studying various human diseases, including SCAP-related disorders that would benefit from reduced SCAP expression. These models can be used for in vivo testing of iRNAs and for determining effective therapeutic doses. Suitable mouse models are known in the art, including, for example, obese (ob / ob) mice containing mutations in the obesity (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); mice containing homozygous knockout of the LDL receptor (LDLR- / - mice; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); diet-induced atherosclerosis mouse models (Ishida et al., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoprotein lipase knockout mouse models (Weistock et al., (1995) J. Clin. Invest. 96(6):2555-2568).

[0337] 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 a powder or aerosol, such as by a nebulizer; intratracheal, intranasal, transepidermal and transdermal, oral or parenteral. Parenteral administration may be 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.

[0338] iRNAs can be delivered in a manner that targets specific tissues, such as the liver (e.g., hepatic parenchymal cells).

[0339] 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 and gloves may also be useful. Suitable topical formulations include those in which the iRNA addressed in the present 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, distearolyphosphatidyl choline), 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 arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Examples include, but are not limited to, 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, which is incorporated herein by reference.

[0340] 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 that of biological membranes, 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 RNAi. In some cases, liposomes are also specifically targeted, for example, to induce iRNAs into specific cell types.

[0341] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed 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 RNAi agent preparation is added to the micelles containing the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain the RNAi agent liposome preparation.

[0342] If necessary, a support compound that promotes condensation may 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). The pH may also be adjusted to support condensation.

[0343] 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 in Felgner, PLet al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; al.,(1979)Biochim.Biophys.Acta 557:9;Szoka et al.,(1978)Proc.Natl.Acad.Sci.75:4194;Mayhew et al.,(1984)Biochim.Biophys.Acta 775:169;Kim et al.,(1983)Biochim.Biophys.Acta 728:339; and Fukunaga et al. This may also include one or more embodiments of the exemplary methods described in al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and combinations of freeze-thaw and extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161). Micro-solution preparation may be used when consistently small (50–200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are readily adaptable to the packing of RNAi preparations into liposomes.

[0344] 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 complex binds to the negatively charged cell surface and is taken into the endosome. Due to the acidic pH inside the endosome, the liposome ruptures, releasing its contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0345] 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. (1992) Journal of Controlled Release, 19:269-274).

[0346] One major type of liposome composition contains phospholipids in addition to naturally derived phosphatidylcholine. For example, neutral liposome compositions may be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic 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.

[0347] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; U.S. Patent Nos. 5,171,678; International Publication No. 94 / 00569; International Publication No. 93 / 24640; International Publication No. 91 / 16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

[0348] 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., (1994) STPPharma.Sci., 4(6):466).

[0349] 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 circulating lifespan compared to liposomes lacking such specialized lipids. An example of a stereostabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome contains (A) monosialoganglioside G M1(B) These contain one or more glycolipids, or are derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While we do not wish to be constrained by any particular theory, in the art, in sterically stabilized liposomes containing at least 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., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0350] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NYAcad. Sci., (1987), 507:64) described monosialoganglioside G M1 The ability of galactocerebroside sulfate and phosphatidylinositol to improve the half-life of liposomes in the blood has been reported. 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., describe (1) sphingomyelin and (2) ganglioside G M1 Liposomes comprising or galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimiristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0351] 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 RNAi agents to macrophages.

[0352] 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 RNAi 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.

[0353] 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 RNAi agent delivery (for a description of DOTMA and its use in combination with DNA, see, for example, Felgner, Plet al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417 and U.S. Patent No. 4,897,355).

[0354] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA complexing 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.

[0355] 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).

[0356] Another cationic lipid complex involves lipid derivatization by cholesterol ("DC-Chol") combined with DOPE and formulated into liposomes (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for translocation in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). In certain cell lines, these liposomes containing conjugated cationic lipids 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.

[0357] 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 RNAi agents intracutaneously. In some implementations, liposomes are used to deliver RNAi agents to epidermal cells and to enhance the penetration of RNAi 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., (1992) Journal of Drug Targeting, vol.2, 405-410 and du Plessis et al., (1992) Antiviral Research:18, 259-265; Mannino, R. Jand Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. Rand Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Rand See Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855.

[0358] 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. 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, including RNAi agents, are useful for treating skin diseases.

[0359] Liposomes containing iRNA can be highly deformable. Such deformability can allow the liposome to penetrate pores smaller than the average radius of the liposome. For example, transferosomes are a type of deformable liposome. Transferosomes can be created by adding surface edge activators, usually surfactants, to a standard liposome composition. Transferosomes containing RNAi agents can be delivered subcutaneously, for example by infection, to deliver the RNAi 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.

[0360] Other formulations to which the present invention can be applied are described in U.S. Provisional Patent Application No. 61 / 018,616, filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed on March 26, 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed on April 22, 2008; and U.S. Provisional Patent Application No. 61 / 051,528, filed on May 8, 2008. PCT Application PCT / US2007 / 080331, filed on October 3, 2007, also describes formulations to which the present invention can be applied.

[0361] 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 their targets without fragmentation and self-load. 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.

[0362] 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 surfactant types, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0363] 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 common members of the nonionic surfactant class.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] The use of surfactants in pharmaceuticals, formulations, and emulsions is outlined (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0368] 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.

[0369] Mixed micelle formulations suitable for transdermal delivery include siRNA compositions and alkali metals C8-C8. 22 The mixture may be prepared by mixing an aqueous solution of an alkyl sulfate and 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 substantially, but they are mixed vigorously to provide smaller micelles.

[0370] 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.

[0371] 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.

[0372] 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 become 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.

[0373] 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.

[0374] 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 via the gastrointestinal tract.

[0375] B. Lipid particles For example, iRNAs such as the dsRNA of the present invention may be completely encapsulated in a lipid formulation such as LNP or other nucleic acid-lipid particles.

[0376] As used herein, the term “LNP” refers to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid complexes). LNPs exhibit a long circulatory lifetime following intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic applications. Examples of LNPs include “pSPLP,” which contains an encapsulation condenser-nucleic acid complex 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.

[0377] In one embodiment, the ratio of lipids to drugs (mass / mass ratio) (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.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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.

[0382] Conjugated lipids that inhibit particle aggregation may include, for example, polyethylene glycol (PEG) lipids, including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, 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.

[0383] 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.

[0384] 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, the contents of which are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Each stock solution in ethanol may be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 may then be combined in a molar ratio, for example, 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) so that the final ethanol concentration is approximately 35-45% and the final sodium acetate concentration is approximately 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]

[0385] The LNP01 formulation is described, for example, in International Publication No. 2008 / 042973, which is incorporated herein by reference.

[0386] Further exemplary lipid-dsRNA preparations are listed in the table below.

[0387] [Table 1-1]

[0388] [Table 1-2]

[0389] 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.

[0390] Formulations containing XTC are described in International Publication No. 2010 / 088537 (all of which are incorporated herein by reference).

[0391] 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.

[0392] Formulations containing ALNY-100 are described in International Publication No. 2010 / 054406 (all of which are incorporated herein by reference).

[0393] Formulations containing C12-200 are described in International Publication No. 2010 / 129709 (all of which are incorporated herein by reference).

[0394] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, suspensions or solutions in water or non-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 DsRNAs 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, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexyl Examples include acrylates, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, methyl polyacrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginates, 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.

[0395] Compositions and formulations for parenteral, intracerebral (intracerebral), subarachnoid, intraventricular, or intrahepatic administration may include sterile aqueous solutions, which may also include buffers, diluents, and other suitable additives, including but not limited to osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0396] 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.

[0397] 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 includes 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 as needed.

[0398] 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.

[0399] 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 the dispersed phase and the active agent, which may exist as a solution in either the 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, which contain more than two phases. Such complex formulations often offer certain 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 in a continuous oily phase provides an o / w / o emulsion.

[0400] Emulsions are characterized by 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).

[0401] 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 hydrophilicity to hydrophobicity 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).

[0402] 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.

[0403] 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).

[0404] 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.

[0405] 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. 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.

[0406] 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.

[0407] 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 system of water, oil, and amphiphilic substances that is a single optically isotropic and thermodynamically stable solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, 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).

[0408] 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.

[0409] 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 typically be, but is not limited to, water, aqueous solutions of pharmaceuticals, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0410] 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.

[0411] 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. The 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.

[0412] iii. Particulates The RNAi agent of the present invention may be incorporated into particles, such as microparticles. The microparticles may 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.

[0413] 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 crossed is treated with a penetration enhancer. In addition to promoting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0414] 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.

[0415] 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).

[0416] 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, and its C 1~20 Examples include 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).

[0417] 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).(For example, see 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).

[0418] 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). (For example, see 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).

[0419] 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).

[0420] Substances that enhance iRNA uptake at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids such as lipofectin (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 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), RNAiMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent(Roche;Grenzacherstrasse,Switzerland), DOTAP Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), DOSPER Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), or Fugene(Grenzacherstrasse,Switzerland), Transfectam(R) Reagent(Promega;Madison,WI), TransFast(TM)Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass.a D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM) / LipoGen(TM) (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER(TM) transfection Reagent (Genlantis; San Examples include RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect (trademark) (B-Bridge International, Mountain View, CA, USA).

[0421] Other active ingredients, including glycols such as ethylene glycol and propylene glycol; pyrroles such as 2-pyrrole; azone; and terpenes such as limonene and menthone, can be used to enhance the penetration of administered nucleic acids.

[0422] 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 its analogue that is inactive (i.e., not biologically active itself) but is recognized as a nucleic acid by an in vivo process, for example, by degrading a biologically active nucleic acid or facilitating its 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 the 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).

[0423] 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).

[0424] 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.

[0425] 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 excipi...

Claims

1. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of sterol regulatory element-binding protein (SREBP) chaperone (SCAP) genes, comprising a sense strand and an antisense strand, The sense strand comprises at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NOs. 1 to 13 by three nucleotides or less; A double-stranded RNAi agent wherein the antisense strand contains at least 15 consecutive nucleotides that differ by three nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 14 to 26.

2. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of sterol regulatory element-binding protein (SREBP) chaperone (SCAP) genes, comprising a sense strand and an antisense strand, A double-stranded RNAi agent wherein the antisense strand includes a complementary region comprising at least 15 consecutive nucleotides that differ by three nucleotides or less from any one of the antisense sequences listed in Tables 2, 3, 5, and 6.

3. The double-stranded RNAi agent according to claim 1 or 2, wherein the sense strand and antisense strand include sequences selected from the group consisting of any one of the sequences in Table 2, Table 3, Table 5, and Table 6.

4. The sense strand consists of nucleotides 345-363, 378-396, 383-401, 402-420, 437-455, 458-476, 491-509, 1014-1032, 1237-1255, 1243-1261, 1259-1277, 1318-1336, 1323-1341, 1497-1515, 1571-1589, 1588-1606, 1605-1623, 1 725-1743, 1767-1785, 1946-1964, 2004-2022, 2160-2178, 2193-2211, 2217-2235, 2517-2535, 2547-2565, 2616-2634, 2663-2681, 2717-2735, 2734-2752, 2751-2769, 2874-2892, 2885-2903, 2998-3016, 3276-329 4, 3292-3310, 3325-3343, 3342-3360, 3420-3438, 3469-3487, 3478-3496, 3533-3551, 3549-3567, 3565-3583, 3579-3597, 3622-3640, 3667-3685, 3771-3789, 3788-3806, 3871-3889, 3891-3909, 3904-3922, 3921- A double-stranded ribonucleic acid (RNAi) agent according to claim 1 or 2, comprising at least 15 consecutive nucleotides that differ by three nucleotides or less from the nucleotide sequences 3939, 4002-4020, 4010-4028, 4027-4045, 4075-4093, 4129-4147, 4145-4163, 4149-4167, 4168-4186, 4184-4202, and 4197-4215.

5. The aforementioned antisense chains are listed in Tables 5 and 6 as follows: AD-77633, AD-77631, AD-77630, AD-77629, AD-77627, AD-77625, AD-77624, AD-77587, AD-77573, AD-77572, AD-77571, AD-77567, AD-77566, AD-77738, AD-77731, AD-77730, AD-77729, AD-77721, AD-77718, AD-77706, AD-77701, AD-77690, AD-77688, AD-77686, AD-77669, AD-77 667, AD-77662, AD-77660, AD-77656, AD-77655, AD-77654, AD-77555, AD-77554, AD-77547, AD-77530, A D-77529, AD-77527, AD-77526, AD-77521, AD-77519, AD-77518, AD-77514, AD-77513, AD-77512, AD-775 10, AD-77507, AD-77505, AD-77499, AD-77498, AD-77494, AD-77492, AD-77491, AD-77490, AD-77486, AD A double-stranded ribonucleic acid (RNAi) agent according to claim 1 or 2, comprising a double-stranded antisense nucleotide sequence selected from the group consisting of -77485, AD-77484, AD-77483, AD-77479, AD-77478, AD-77477, AD-77476, AD-77475, and AD-77474, and at least 15 consecutive nucleotides that differ by three nucleotides or less.

6. A double-stranded RNAi agent according to claim 1 or 2, comprising at least one modified nucleotide.

7. The double-stranded RNAi agent according to any one of claims 1 to 6, wherein substantially all of the nucleotides of the sense strand are modified nucleotides.

8. The double-stranded RNAi agent according to any one of claims 1 to 6, wherein substantially all of the nucleotides of the antisense strand are modified nucleotides.

9. The double-stranded RNAi agent according to any one of claims 1 to 6, wherein all of the nucleotides in the sense strand are modified nucleotides.

10. The double-stranded RNAi agent according to any one of claims 1 to 6, wherein all of the nucleotides in the antisense strand are modified nucleotides.

11. The double-stranded RNAi agent according to any one of claims 1 to 6, wherein all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

12. At least one of the modified nucleotides is a deoxynucleotide, 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 conformationally 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 phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexyl A double-stranded RNAi agent according to any one of claims 6 to 10, selected from the group consisting of xenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 5'-phosphate mimetic, nucleotides containing vinyl phosphate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing a thymidine-glycol nucleic acid (GNA) S isomer, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and cholesteryl derivatives and terminal nucleotides linked to a dodecanoic acid bisdecylamide group.

13. The double-stranded RNAi agent according to claim 12, wherein the modified nucleotide is selected from the group consisting of nucleotides comprising 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymine nucleotides (dT), locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases.

14. The double-stranded RNAi agent according to claim 12, wherein the modified nucleotide includes a short sequence of 3'-terminal deoxythymine nucleotide (dT).

15. The double-stranded RNAi agent according to claim 12, wherein the modification to the nucleotide is a 2'-O-methyl modification and a 2'-fluoro modification.

16. The double-stranded RNAi agent according to claim 12, further comprising at least one phosphorothioate nucleotide interbonding.

17. The double-stranded RNAi agent according to claim 16, comprising 6 to 8 phosphorothioate nucleotide interbondings.

18. The double-stranded RNAi agent according to claim 1 or 2, wherein the complementary region is at least 17 nucleotides long.

19. The double-stranded RNAi agent according to claim 1 or 2, wherein the complementary region is 19 to 23 nucleotides long.

20. The double-stranded RNAi agent according to claim 1 or 2, wherein the complementary region is 19 nucleotides long.

21. A double-stranded RNAi agent according to any one of claims 1 to 20, wherein each strand is 30 nucleotides or less in length.

22. A double-stranded RNAi agent according to claim 1 or 2, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

23. A double-stranded RNAi agent according to claim 1 or 2, wherein at least one strand comprises 3' overhangs of at least two nucleotides.

24. The double-stranded RNAi agent according to claim 1 or 2, further comprising an N-acetylgalactosamine (GalNAc) derivative conjugated to the 3' end of the sense strand via a monovalent or branched divalent or trivalent linker.

25. The ligand is 【Chemistry 1】 The double-stranded RNAi agent according to claim 24.

26. The following schematic diagram 【Chemistry 2】 The double-stranded RNAi agent according to claim 24, which is conjugated to a ligand as shown in the formula, wherein X is O or S.

27. The double-stranded RNAi agent according to claim 26, wherein X is O.

28. The double-stranded RNAi agent according to claim 24, wherein the ligand is cholesterol.

29. The double-stranded RNAi agent according to claim 2, wherein the complementary region comprises one of the antisense sequences in any one of Tables 2, 3, 5, and 6.

30. The double-stranded RNAi agent according to claim 2, wherein the complementary region consists of one of the antisense sequences in any one of Tables 2, 3, 5, and 6.

31. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene, comprising an antisense strand and a sense strand complementary thereto, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, and each strand is approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent comprises formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ ' - N ａ ' - (X'X'X') ｋ - N ｂ ' - Y'Y'Y' - N ｂ ' - (Z'Z'Z') ｌ - N ａ ' - n ｑ '5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; Each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is independently either modified or unmodified, or a combination thereof; each n ｐ , n ｐ ', n ｑ , and n ｑ Each of these may or may not exist, independently representing 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 to three consecutive nucleotides; N ｂ The modification to N is different from the modification to Y, and N ｂ The modifier to ' is expressed by (which is different from the modifier to Y'); A double-stranded RNAi agent in which the sense strand is conjugated to at least one ligand.

32. The double-stranded RNA i agent according to claim 31, 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.

33. The double-stranded RNAi agent according to claim 31, 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.

34. The double-stranded RNAi agent according to claim 31, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

35. The double-stranded RNAi agent according to claim 31, wherein the YYY motif is present at or near the cleavage site of the sense strand.

36. The double-stranded RNAi agent according to claim 31, wherein the Y'Y'Y' motif is located at the 11, 12, and 13 positions of the antisense strand from the 5' end.

37. The double-stranded RNAi agent according to claim 36, wherein Y' is 2'-O-methyl.

38. The above equation (III) is equation (IIIa): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -Y'Y'Y'-N ａ’ -n ｑ’ 5' (IIIa) A double-stranded RNAi agent according to claim 31, as represented by [the specified method].

39. The above equation (III) is equation (IIIb): Sense: 5'n ｐ -N ａ -YYY-Nb-ZZZ-Na-nq3' Antisense: 3'n ｐ’ -N ａ’ -Y'Y'Y'-N ｂ’ -Z'Z'Z'-N ａ’ -n ｑ’ 5' (IIIb) (In the formula, each N ｂ and N ｂ (' represents an oligonucleotide sequence containing 1 to 5 modified nucleotides independently.) A double-stranded RNAi agent according to claim 31, as represented by [the specified method].

40. The above equation (III) is equation (IIIc): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -X'X'X'-N ｂ’ -Y'Y'Y'-N ａ’ -n ｑ '5' (IIIc) (In the formula, each N ｂ and N ｂ (' represents an oligonucleotide sequence containing 1 to 5 modified nucleotides independently.) A double-stranded RNAi agent according to claim 31, as represented by [the specified method].

41. The above equation (III) is equation (IIId): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ｂ -ZZZ-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -X'X'X'-N ｂ’ -Y'Y'Y'-N ｂ’ -Z'Z'Z'-N ａ’ -n ｑ '5' (IIId) (In the formula, each N ｂ and N ｂ (' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each Na and Na' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides.) A double-stranded RNAi agent according to claim 31, as represented by [the specified method].

42. The double-stranded RNAi agent according to claim 31, wherein the double-stranded region is 15 to 30 nucleotide pairs long.

43. The double-stranded RNAi agent according to claim 42, wherein the double-stranded region is 17 to 23 nucleotide pairs long.

44. The double-stranded RNAi agent according to claim 42, wherein the double-stranded region is 17 to 25 nucleotide pairs long.

45. The double-stranded RNAi agent according to claim 42, wherein the double-stranded region is 23 to 27 nucleotide pairs long.

46. The double-stranded RNAi agent according to claim 42, wherein the double-stranded region is 19 to 21 nucleotide pairs long.

47. The double-stranded RNAi agent according to claim 31, wherein the double-stranded region is 21 to 23 nucleotide pairs long.

48. The double-stranded RNAi agent according to claim 31, wherein each strand is 15 to 30 nucleotides long.

49. The double-stranded RNAi agent according to claim 31, wherein each strand is 19 to 30 nucleotides long.

50. The double-stranded RNAi agent according to claim 31, wherein the modification to the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

51. The double-stranded RNAi agent according to claim 50, wherein the modification to the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

52. The double-stranded RNAi agent according to claim 31, wherein the ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched linker; or cholesterol.

53. The ligand is 【Transformation 3】 The double-stranded RNAi agent according to claim 31.

54. The double-stranded RNAi agent according to claim 31, wherein the ligand is bound to the 3' end of the sense strand.

55. The following schematic diagram 【Chemistry 4】 The double-stranded RNAi agent according to claim 54, which is conjugated to the ligand as shown.

56. The double-stranded RNAi agent according to claim 31, further comprising at least one phosphorothioate or methylphosphonate internucleotide bond.

57. The double-stranded RNAi agent according to claim 56, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 3' end of one of the strands.

58. The double-stranded RNAi agent according to claim 57, wherein the strand is the antisense strand.

59. The double-stranded RNAi agent according to claim 57, wherein the strand is the sense strand.

60. The double-stranded RNAi agent according to claim 56, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 5' end of one of the strands.

61. The double-stranded RNAi agent according to claim 60, wherein the strand is the antisense strand.

62. The double-stranded RNAi agent according to claim 60, wherein the strand is the sense strand.

63. The double-stranded RNAi agent according to claim 56, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at both the 5' and 3' ends of one of the strands.

64. The double-stranded RNAi agent according to claim 63, wherein the strand is the antisense strand.

65. The double-stranded RNAi agent according to claim 31, wherein the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair.

66. The double-stranded RNAi agent according to claim 31, wherein the Y nucleotide contains a 2'-fluoro modification.

67. The double-stranded RNAi agent according to claim 31, wherein the Y' nucleotide contains a 2'-O-methyl modification.

68. The double-stranded RNAi agent according to claim 31, wherein p' > 0.

69. The double-stranded RNAi agent according to claim 31, wherein p' = 2.

70. The double-stranded RNAi agent according to claim 69, wherein q'=0, p=0, q=0, and the p' overhang nucleotide is complementary to the target mRNA.

71. The double-stranded RNA i agent according to claim 69, wherein q' = 0, p = 0, q = 0, and the p' overhang nucleotide is non-complementary to the target mRNA.

72. The double-stranded RNAi agent according to claim 63, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

73. at least one n ｐ A double-stranded RNAi agent according to any one of claims 68 to 72, wherein ' is linked to an adjacent nucleotide via a phosphorothioate bond.

74. All n ｐ The double-stranded RNAi agent according to claim 73, wherein ' is linked to an adjacent nucleotide via a phosphorothioate bond.

75. A double-stranded RNAi agent according to claim 31, selected from the group of RNAi agents listed in either Table 2 or Table 3.

76. The double-stranded RNAi agent according to claim 31, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified.

77. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, comprising an antisense strand and a sense strand complementary thereto, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, and each strand is approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent comprises formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1; p, p', q, and q' are each independently between 0 and 6; Each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' each independently represents an oligonucleotide sequence comprising 0 to 10 nucleotides, which may be either modified or unmodified, or a combination thereof; each n ｐ , n ｐ ', n ｑ , and n ｑ Each of these may or may not exist, independently representing 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 to three consecutive nucleotides, wherein the modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N ｂ The modification to N, unlike the modification to Y, and ｂ the modification to N’ which is different from the modification to Y’ is represented by; A double-stranded RNAi agent in which the sense strand is conjugated to at least one ligand.

78. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, comprising an antisense strand and a sense strand complementary thereto, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, and each strand is approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent comprises formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1; each n ｐ , n ｑ , and n ｑ Each of these may or may not exist, independently representing an overhang nucleotide; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications to three consecutive nucleotides, wherein the modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N ｂ The modification to N is different from the modification to Y, and N ｂ The modifier to ' is expressed by (which is different from the modifier to Y'); A double-stranded RNAi agent in which the sense strand is conjugated to at least one ligand.

79. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, comprising an antisense strand and a sense strand complementary thereto, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, and each strand is approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent comprises formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1; each n ｐ , n ｑ , and n ｑ Each of these may or may not exist, independently representing an overhang nucleotide; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications to three consecutive nucleotides, wherein the modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N ｂ The modification to N is different from the modification to Y, and N ｂ The modifier to ' is expressed by (which is different from the modifier to Y'); A double-stranded RNAi agent in which the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

80. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, comprising an antisense strand and a sense strand complementary thereto, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, and each strand is approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent comprises formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula, i, j, k, and l are each independently either 0 or 1; each n ｐ , n ｑ , and n ｑ Each of these may or may not exist, independently representing an overhang nucleotide; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which is independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications to three consecutive nucleotides, wherein the modifications are 2'-O-methyl modifications or 2'-fluoro modifications; N ｂ The modification to N is different from the modification to Y, and N ｂ The modifier to ' is expressed by (which is different from the modifier to Y'); The sense chain comprises at least one phosphorothioate bond; A double-stranded RNAi agent in which the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

81. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the sterol regulatory element-binding protein (SREBP) chaperone (SCAP) gene in cells, comprising an antisense strand and a sense strand complementary thereto, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding SCAP, and each strand is approximately 14 to approximately 30 nucleotides long, and the double-stranded RNAi agent comprises formula (III): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-Y'Y'Y'-N ａ '-n ｑ '5' (IIIa) (In the formula, each n ｐ , n ｑ , and n ｑ Each of these may or may not exist, independently representing an overhang nucleotide; p, q, and q' are each independently between 0 and 6; n ｐ '>0 and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, which is either modified or unmodified or a combination thereof, and each sequence contains at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent one motif of three identical modifications to three consecutive nucleotides, the modifications being either 2'-O-methyl or 2'-fluoro modifications; The sense chain comprises at least one phosphorothioate bond; A double-stranded RNAi agent in which the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

82. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of sterol regulatory element-binding protein (SREBP) chaperone (SCAP) genes, It includes a sense strand and an antisense strand that form a double-stranded region, The sense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 1 to 13, and the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 14 to 26. Substantially all of the nucleotides of the sense strand include modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications. The sense strand includes two phosphorothioate nucleotide interlinks at its 5' end. Substantially all of the nucleotides of the antisense strand include modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications. The antisense strand includes two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end. A double-stranded RNAi agent in which the sense strand is conjugated to one or more GalNAc derivatives linked at the 3' end via a branched divalent or trivalent linker.

83. A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of sterol regulatory element-binding protein (SREBP) chaperone (SCAP) genes, It includes a sense strand and an antisense strand that form a double-stranded region, The sense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 1 to 13, and the antisense strand comprises at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the nucleotide sequences of SEQ ID NOs. 14 to 26. The sense strand comprises at least one 3'-terminal deoxythymine nucleotide (dT), A double-stranded RNAi agent wherein the antisense strand contains at least one 3' terminal deoxythymine nucleotide (dT).

84. The double-stranded RNAi agent according to claim 82, wherein all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand contain modified nucleotides.

85. A double-stranded RNAi agent according to claim 82 or 83, wherein each strand has 19 to 30 nucleotides.

86. Cells containing a double-stranded RNAi agent according to any one of claims 1 to 85.

87. A pharmaceutical composition for inhibiting the expression of a SCAP gene, comprising a double-stranded RNAi agent according to any one of claims 1 to 85.

88. The pharmaceutical composition according to claim 87, wherein the double-stranded RNAi agent is administered in a non-buffer solution.

89. The pharmaceutical composition according to claim 88, wherein the non-buffer solution is physiological saline or water.

90. The pharmaceutical composition according to claim 87, wherein the double-stranded RNAi agent is administered using a buffer solution.

91. The pharmaceutical composition according to claim 90, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

92. The pharmaceutical composition according to claim 90, wherein the buffer solution is phosphate-buffered saline (PBS).

93. A pharmaceutical composition comprising a double-stranded RNAi agent according to any one of claims 1 to 85 and a lipid preparation.

94. The pharmaceutical composition according to claim 93, wherein the lipid preparation contains LNP.

95. The pharmaceutical composition according to claim 93, wherein the lipid preparation comprises MC3.

96. A method for inhibiting the expression of sterol regulatory element-binding protein (SREBP) chaperone (SCAP) genes in cells, (a) The step of bringing the cells into contact with the double-stranded RNAi agent according to any one of claims 1 to 85 or the pharmaceutical composition according to any one of claims 87 to 95; (b) The step of maintaining the cells generated in step (a) for a sufficient time to achieve degradation of the mRNA transcript of the SCAP gene, thereby inhibiting the expression of the SCAP gene in the cells. A method that includes this.

97. The method according to claim 96, wherein the cells are located within the target area.

98. The method according to claim 97, wherein the subject is a human.

99. The method according to claim 97, wherein the subject is selected from the group consisting of rhesus macaques, cynomolgus macaques, mice, and rats.

100. The method according to claim 98, wherein the human subject is suffering from SCAP-related disorder.

101. The method according to claim 100, wherein the SCAP-related disease is non-alcoholic fatty liver disease (NAFLD).

102. The method according to claim 100, wherein the SCAP-related disorder is fatty liver (steatosis).

103. The method according to claim 100, wherein the SCAP-related disorder is non-alcoholic steatohepatitis (NASH).

104. The method according to any one of claims 96 to 103, wherein SCAP expression is inhibited by at least about 30%.

105. A method for treating a subject having a disorder that would benefit from reduced SCAP expression, comprising the step of administering to the subject a therapeutically effective amount of a double-stranded RNAi agent according to any one of claims 1 to 85 or a pharmaceutical composition according to any one of claims 87 to 95, thereby treating the subject.

106. The method according to claim 105, wherein the subject is suffering from SCAP-related disorder.

107. The method according to claim 105, wherein the subject is a human.

108. The method according to claim 106, wherein the SCAP-related disease is non-alcoholic fatty liver disease (NAFLD).

109. The method according to claim 106, wherein the SCAP-related disease is fatty liver (steatosis).

110. The method according to claim 106, wherein the SCAP-related disease is non-alcoholic steatohepatitis (NASH).

111. The method according to any one of claims 105 to 110, wherein SCAP expression is inhibited by at least about 30%.

112. The method according to any one of claims 105 to 111, further comprising the step of administering an additional therapeutic agent to the subject.

113. The method according to any one of claims 105 to 112, wherein the double-stranded RNAi agent is administered in a dose of about 0.01 mg / kg to about 50 mg / kg.

114. The method according to any one of claims 105 to 113, wherein the double-stranded RNAi agent is administered subcutaneously to the subject.

115. The method according to any one of claims 105 to 114, wherein the administration of the double-stranded RNAi to the subject causes a decrease in one or more serum lipids and a decrease in SCAP protein accumulation.

116. The method according to any one of claims 105 to 115, wherein the administration of the dsRNA to the subject causes a decrease in PNPLA3 protein accumulation or SREBP double-stranded RNAi accumulation.

117. A method for inhibiting SCAP expression in a subject, comprising the step of administering a therapeutically effective amount of a double-stranded RNAi agent according to any one of claims 1 to 85 or a pharmaceutical composition according to any one of claims 87 to 95 to the subject, thereby inhibiting the expression of SCAP in the subject.

118. A method for reducing plasma triglyceride levels in a subject, comprising the step of administering a therapeutically effective amount of a double-stranded RNAi agent according to any one of claims 1 to 85 or a pharmaceutical composition according to any one of claims 87 to 95 to the subject, thereby reducing the plasma triglyceride levels in the subject.

119. A method for inhibiting the progression of NAFLD in a subject, comprising the step of administering a therapeutically effective amount of a double-stranded RNAi agent according to any one of claims 1 to 85 or a pharmaceutical composition according to any one of claims 87 to 95 to the subject, thereby inhibiting the progression of NAFLD in the subject.

120. A kit for carrying out the method described in any one of claims 105 or 117 to 119, a) The double-stranded RNAi agent and, b) Instructions for use and c) A means of administering the double-stranded RNAi agent to the subject, at the discretion of the subject. A kit that includes this.

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