Patatin-like phospholipase domain-containing 3(PNPLA3)iRNA composition and method of use thereof

Abstract

To provide therapies for subjects suffering from NAFLD.SOLUTION: A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of Patatin-Like Phospholipase Domain Containing 3 is used which comprises a sense strand and an antisense strand. The sense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from a specific nucleotide sequence. The antisense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from another specific nucleotide sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 115,724, filed on 13 February 2015, and to U.S. Provisional Patent Application No. 62 / 266,818, filed on 14 December 2015. The aforementioned 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, prepared on 11 February 2016, is named 121301-03220_SL.txt and has a size of 529,799 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] In 2008, a genome-level association study of the liver using proton magnetic resonance spectroscopy to assess liver fat content revealed a significant association between liver fat content and the patatin-like phospholipase domain 3 (PNPLA3) gene (see, for example, Non-Patent Literature 1). Studies using knock-in mice revealed that expression of a sequence polymorphism in PNPLA3 (rs738409, I148M) induces NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surface of lipid droplets is associated with hepatic triglyceride accumulation (Non-Patent Literature 2). In particular, the PNPLA3 I148M mutant is associated with promoting fibrillation by activating the Hedgehog (Hh) signaling pathway, resulting in activation and proliferation of hepatic stellate cells and deposition of extracellular matrix (Non-Patent Literature 3). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Romeo et al. (2008) Nat. Genet.,40(12):1461-1465 [Non-Patent Document 2] Smagris et al.(2015)Hepatology,61:108-118 [Non-Patent Document 3] Chen et al.(2015)World J.Gastroenterol.,21(3):794-802 [Overview of the project] [Problems that the invention aims to solve]

[0007] Currently, treatment for NAFLD focuses on weight loss and the management of secondary conditions such as insulin resistance or dyslipidemia. To date, no pharmacological treatments for NAFLD have been approved. Therefore, there is a need for treatment options for individuals suffering from NAFLD. [Means for solving the problem]

[0008] The present invention provides an iRNA composition that induces RNA-induced silencing complex (RISC) mediated cleavage of the RNA transcript of the PNPLA3 gene. The PNPLA3 gene may be present in cells, such as those in a subject such as a human cell. [Brief explanation of the drawing]

[0009] [Figure 1] This graph shows the percentage of PNPLA3 mRNA remaining in the liver of ob / ob mice after administration of a single dose of the iRNA agent shown, at 0.3 mg / kg, 1.5 mg / kg, or 3.0 mg / kg. [Modes for carrying out the invention]

[0010] In one embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of patatin-like phospholipase domain 3 (PNPLA3), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprising at least 15 consecutive nucleotides that differ by 3 nucleotides or less from the nucleotide sequence of SEQ ID NO: 2.

[0011] In one embodiment, the sense strand and the antisense strand include sequences selected from the group consisting of any one of the sequences in Tables 3-5, 7, and 8.

[0012] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of patatin-like phospholipase domain 3 (PNPLA3), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region containing at least 15 consecutive nucleotides that differ by 3 nucleotides or less from any one of the antisense sequences listed in any one of Tables 3-5, 7, and 8.

[0013] In one embodiment, the double-stranded RNAi agent contains at least one modified nucleotide. In another embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

[0014] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of patatin-like phospholipase domain 3 (PNPLA3), 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 differing by 3 or less nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprising at least 15 consecutive nucleotides differing by 3 or less nucleotides from the nucleotide sequence of SEQ ID NO: 2, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand is conjugated to a ligand attached to its 3' end.

[0015] In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 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, debasalized 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, non-natural base-containing nucleotides, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, phosphorothioate-containing nucleotides, methylphosphonate-containing nucleotides, 5'-phosphate-containing nucleotides, and 5'-phosphate mimetic-containing nucleotides. In another embodiment, the modified nucleotide includes a short sequence of 3'-terminal deoxythymine nucleotides (dT).

[0016] In one embodiment, the complementary region is at least 17 nucleotides long. In another embodiment, the complementary region is 19 to 21 nucleotides long. In yet another embodiment, the complementary region is 19 nucleotides long. In yet another embodiment, each chain is 30 nucleotides or less long.

[0017] In one embodiment, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides.

[0018] In one embodiment, the double-stranded RNAi agent further comprises a ligand. In one embodiment, the ligand conjugates to the 3'-end of the sense strand of the double-stranded RNAi agent. In another embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In one embodiment, the ligand is [Chemical formula] as follows.

[0019] In another embodiment, the double-stranded RNAi agent has the following schematic diagram: [Chemical formula] conjugated to the ligand shown in the formula, where X is O or S. In one embodiment, X is O.

[0020] In one embodiment, the complementary region comprises one of the antisense sequences of any one of Tables 3-5, 7, and 8. In another embodiment, the complementary region consists of one of the antisense sequences of any one of Tables 3-5, 7, and 8.

[0021] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, the antisense strand comprises a region complementary to a part of the mRNA encoding PNPLA3, and each strand is about 14 to about 30 nucleotides in length, wherein 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) It is represented by, 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; and each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, each independently being either modified or unmodified or a combination thereof, where 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 are independently modified, 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 Modifications to N are different from modifications to Y. b The modification to ' is different from the modification to Y'; here, the sense strand is conjugated to at least one ligand.

[0022] In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1. In another embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'. In another embodiment, the YYY motif is located at or near the cleavage site of the sense chain. In another embodiment, the Y'Y'Y' motif is located at positions 11, 12, and 13 of the antisense chain from the 5' end. In one embodiment, Y' is 2'-O-methyl.

[0023] In one embodiment, 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].

[0024] In another embodiment, 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) It is represented by, In the formula, each N b and N b The '' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0025] In another embodiment, 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) It is represented by, In the formula, each N b and N b The '' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0026] In another embodiment, 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) It is represented by, In the formula, each N b and N b ' represents an oligonucleotide sequence containing 1 to 5 modified nucleotides independently, each N a and N a ' represents an oligonucleotide sequence containing 2 to 10 modified nucleotides independently.

[0027] In one embodiment, the double-stranded region is 15 to 30 nucleotide pairs long. In another embodiment, the double-stranded region is 17 to 23 nucleotide pairs long. In yet another embodiment, the double-stranded region is 17 to 25 nucleotide pairs long. In yet another embodiment, the double-stranded region is 23 to 27 nucleotide pairs long. In yet another embodiment, the double-stranded region is 19 to 21 nucleotide pairs long. In yet another embodiment, the double-stranded region is 21 to 23 nucleotide pairs long.

[0028] In one embodiment, each chain has 15 to 30 nucleotides. In another embodiment, each chain has 19 to 30 nucleotides.

[0029] In one embodiment, 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 another embodiment, the modification to the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0030] In one embodiment, the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker. In one embodiment, the ligand is [ka] That is the case.

[0031] In one embodiment, the ligand binds to the 3' end of the sense strand.

[0032] In one embodiment, the double-stranded RNAi agent is shown in the following schematic diagram. [ka] As shown, it is conjugated to a ligand.

[0033] In one embodiment, the double-stranded RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide bond. In one embodiment, the phosphorothioate or methylphosphonate internucleotide bond is located at the 3' end of one of the strands. In one embodiment, this strand is the antisense strand. In another embodiment, this strand is the sense strand.

[0034] In one embodiment, the phosphorothioate or methylphosphonate internucleotide bond is located at the 5' end of one of the strands. In another embodiment, this strand is an antisense strand. In yet another embodiment, this strand is a sense strand.

[0035] In one embodiment, the phosphorothioate or methylphosphonate internucleotide bond is located at both the 5' and 3' ends of one of the strands. In another embodiment, this strand is an antisense strand.

[0036] In one embodiment, the base pair at position 1 of the 5' end of the double-stranded antisense strand is an AU base pair.

[0037] In one embodiment, the Y nucleotide includes a 2'-fluoro modification. In another embodiment, the Y' nucleotide includes a 2'-O-methyl modification. In another embodiment, p'>0. In another embodiment, p'=2. In another embodiment, q'=0, p=0, q=0, and the p' overhang nucleotide is complementary to the target mRNA. In another embodiment, q'=0, p=0, q=0, and the p' overhang nucleotide is incomplementary to the target mRNA.

[0038] In one embodiment, the sense strand has a total of 21 nucleotides, and the antisense strand has a total of 23 nucleotides.

[0039] In one embodiment, at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond. In another embodiment, all n p’ is linked to an adjacent nucleotide via a phosphorothioate linkage.

[0040] In one embodiment, the double-stranded RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3-5, 7, and 8. In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain modifications.

[0041] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in a cell, 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 PNPLA3, each strand is about 14 to about 30 nucleotides in length, and wherein 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) is represented by, wherein 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 comprising from 0 to 25 nucleotides, either modified or unmodified or a combination thereof, each sequence containing 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 independently and each represents an overhang nucleotide; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent one motif of three identical modifications to three consecutive nucleotides, and the modification is a 2’-O-methyl or 2’-fluoro modification; the modification to N b is different from the modification to Y, and the modification to N b ' is different from the modification to Y’; here, the sense strand is conjugated to at least one ligand.

[0042] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3 in cells, where the double-stranded RNAi agent includes a sense strand complementary to the antisense strand, the antisense strand includes a region complementary to a part of the mRNA encoding PNPLA3, each strand is about 14 to about 30 nucleotides in length, and here, 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) is represented by, wherein i, j, k, and l are each independently 0 or 1; each n p, n q , and n q Each of these may or may not exist, and independently represents 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 adjacent nucleotides via phosphorothioate bonds; each N a and N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, each independently being either modified or unmodified or a combination thereof, where 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 are 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, where the modifications are 2'-O-methyl or 2'-fluoro modifications; N b Modifications to N are different from modifications to Y. b The modification to ' is different from the modification to Y'; here, the sense strand is conjugated to at least one ligand.

[0043] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting PNPLA3 expression 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 PNPLA3, and each strand being approximately 14 to approximately 30 nucleotides long, wherein 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) It is represented by, In the formula, i, j, k, and l are each independently either 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, each independently being either modified or unmodified or a combination thereof, where 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 are 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, where the modifications are 2'-O-methyl or 2'-fluoro modifications; N b Modifications to N are different from modifications to Y. b Modification to ' is different from modification to Y'; here, the sense chain is conjugated to at least one ligand, which is one or more GalNAc derivatives bound via a divalent or trivalent branched linker.

[0044] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting PNPLA3 expression 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 PNPLA3, and each strand being approximately 14 to approximately 30 nucleotides long, wherein 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) It is represented by, In the formula, i, j, k, and l are each independently either 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, each independently being either modified or unmodified or a combination thereof, where 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 are 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, where the modifications are 2'-O-methyl or 2'-fluoro modifications; N b Modifications to N are different from modifications to Y. b The modification to ' is different from the modification to Y'; where the sense chain comprises at least one phosphorothioate bond; where 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.

[0045] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting PNPLA3 expression 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 PNPLA3, and each strand being approximately 14 to approximately 30 nucleotides long, wherein 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) It is represented by, 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 aand N a ' represents an oligonucleotide sequence containing 0 to 25 nucleotides, each independently being either modified or unmodified or a combination thereof, where each sequence contains nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent a single motif of three identical modifications to three consecutive nucleotides, where the modifications are 2'-O-methyl or 2'-fluoro modifications; where the sense strand contains at least one phosphorothioate bond; where 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.

[0046] In another embodiment, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PNPLA3, 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 or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprising at least 15 consecutive nucleotides that differ by 3 or fewer nucleotides from the nucleotide sequence of SEQ ID NO: 2, wherein substantially all of the nucleotides of the sense strand are 2'-O-methyl modified and 2'-fluoro modified. The sense strand comprises modifications selected from the group consisting of modifications, with two phosphorothioate nucleotide interlinks at its 5' end, substantially all of the nucleotides of the antisense strand comprises modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, the antisense strand comprises two phosphorothioate nucleotide interlinks at its 5' end and two phosphorothioate nucleotide interlinks at its 3' end, and the sense strand is conjugated at its 3' end to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0047] In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides. In another embodiment, each strand has 19 to 30 nucleotides.

[0048] In another aspect, the present invention provides cells containing a double-stranded RNAi agent, as described herein.

[0049] In another embodiment, the present invention provides a vector encoding at least one strand of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a complementary region to at least a portion of the mRNA encoding PNPLA3, the double-stranded RNAi agent is 30 base pairs or less in length, and the double-stranded RNAi agent targets the mRNA for cleavage. In one embodiment, the complementary region is at least 15 nucleotides long. In another embodiment, the complementary region is 19 to 21 nucleotides long.

[0050] In another aspect, the present invention provides cells comprising the vector described herein.

[0051] In another embodiment, the present invention provides a pharmaceutical composition comprising a double-stranded RNAi agent for inhibiting the expression of the PNPLA3 gene. In one embodiment, the double-stranded RNAi agent is administered in a non-buffered solution. In another embodiment, the non-buffered solution is saline or water. In another embodiment, the double-stranded RNAi agent is administered using a buffer. In another embodiment, the buffer comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffer is phosphate-buffered saline (PBS).

[0052] In another embodiment, the present invention provides a pharmaceutical composition comprising the double-stranded RNAi agent and a lipid preparation. In one embodiment, the lipid preparation comprises LNP. In another embodiment, the lipid preparation comprises MC3.

[0053] In another embodiment, the present invention provides a method for inhibiting the expression of PNPLA3 in cells, the method comprising the steps of (a) contacting cells with a double-stranded RNAi agent or pharmaceutical composition of the present invention; and (b) maintaining the cells produced in step (a) for a time sufficient to achieve degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting the expression of the PNPLA3 gene in the cells. In one embodiment, the cells are within a subject. In another embodiment, the subject is a human. In one embodiment, the subject is a female human. In another embodiment, the subject is a male human. In one embodiment, the expression of PNPLA3 is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0054] In another embodiment, the present invention provides a method for treating a subject having a disease or disorder that would benefit from reduced PNPLA3 expression, the method comprising the step of administering a therapeutically effective amount of the double-stranded RNAi agent or pharmaceutical composition of the present invention to the subject, thereby treating the subject.

[0055] In another embodiment, the present invention provides a method for preventing at least one symptom of a subject having a disease or disorder that benefits from reduced PNPLA3 expression, the method comprising administering a preventive amount of the double-stranded RNAi agent or pharmaceutical composition of the present invention to the subject, thereby preventing at least one symptom of the subject having a disorder that benefits from reduced PNPLA3 expression.

[0056] In one embodiment, administration of a double-stranded RNAi agent to a subject reduces the Hedgehog signaling pathway.

[0057] In one embodiment, PNPLA3-related disease is PNPLA3-related disease. In another embodiment, PNPLA3-related disease is non-alcoholic fatty liver disease (NAFLD). In another embodiment, PNPLA3-related disease is fatty liver (steatosis). In another embodiment, PNPLA3-related disease is non-alcoholic steatohepatitis (NASH). In another embodiment, PNPLA3-related disease is obesity. In one embodiment, the subject is human. In one embodiment, the subject is a female human. In another embodiment, the subject is a male human. In one embodiment, the subject has the PNPLA3 I148M mutation. In one embodiment, the mutation is heterozygous. In another embodiment, the mutation is isozygous.

[0058] In another embodiment, the present invention further includes the step of administering an anti-PNPLA3 antibody or its antigen-binding fragment to a target.

[0059] In one embodiment, the double-stranded RNAi agent is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg or approximately 0.5 mg / kg to approximately 50 mg / kg. In one embodiment, the dsRNA agent is administered in doses of approximately 10 mg / kg to approximately 30 mg / kg. In another embodiment, the dsRNA agent is administered in doses selected from the group consisting of 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg.

[0060] In one embodiment, the double-stranded RNAi agent is administered to the subject once a week. In another embodiment, the double-stranded RNAi agent is administered to the subject once a month.

[0061] In one embodiment, the double-stranded RNAi agent is administered subcutaneously to the subject.

[0062] In another embodiment, the method of the present invention further includes the step of measuring the level of the Hedgehog signaling pathway in a subject. In one embodiment, a decrease in the level of expression or activity of the Hedgehog (Hh) signaling pathway indicates that PNPLA3-related disease is being treated or prevented.

[0063] The present invention will be further described in detail by the following detailed description and drawings.

[0064] Detailed description of the invention The present invention provides iRNA compositions that result in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the patatin-like phospholipase domain-containing gene 3 (PNPLA3). This gene may be present in cells, for example, in human cells. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (PNPLA3 gene) in mammals.

[0065] The RNAi agents of the present invention are designed to target the human PNPLA3 gene, including a portion of the gene conserved within PNPLA3 orthologs of other mammalian species. While we do not wish to impose theoretical constraints, it is believed that the aforementioned properties, as well as specific target portions and / or specific combinations or partial combinations of modifications in these RNAi agents, confer improved efficacy, stability, potency, persistence, and stability to the RNAi agents of the present invention.

[0066] Accordingly, the present invention provides a method for treating subjects with PNPLA3-related diseases, such as non-alcoholic fatty liver disease (NAFLD), that benefit from inhibition or reduction of PNPLA3 gene expression, using an iRNA composition that results in RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PNPLA3 gene.

[0067] In particular, very low doses of the iRNA of the present invention can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of the expression of the corresponding gene (PNPLA3 gene).

[0068] Examples of iRNAs used in this invention include 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, and 19-22. Examples include RNA strands (antisense strands) having regions of approximately 30 nucleotides or less in length, such as 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, and these regions are substantially complementary to at least a portion of the mRNA transcript of the PNPLA3 gene.

[0069] The following detailed description discloses methods for preparing and utilizing compositions containing iRNAs that inhibit the expression of the angiotensinogen gene, as well as compositions, uses, and methods for treating subjects with diseases and disorders that would benefit from the inhibition and / or reduction of the expression of the PNPLA3 gene.

[0070] I. Definition To make the present invention easier to understand, certain terms are defined first. In addition, it should be noted that whenever parameter values ​​or ranges of values ​​are enumerated, intermediate values ​​and ranges of the enumerated values ​​are also intended to be part of the present invention.

[0071] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical purposes of the article. For example, "an element" means one factor or two or more factors, such as multiple factors.

[0072] The term "including" is used herein to mean "including, but not limited to," and is used without distinction from that term.

[0073] 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.

[0074] 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.

[0075] 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 logically consistent with 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 modify each of the numbers within the set of numbers or ranges.

[0076] In the use of this specification, the term "patatin-like phospholipase domain-containing 3," as used interchangeably with the term "PNPLA3," refers to a naturally occurring gene encoding a triacylglycerol lipase that mediates triacylglycerol hydrolysis in adipocytes. The amino acid and complete coding sequences of the reference sequence of the human PNPLA3 gene can be found, for example, in GenBank acceptance number GI:17196625 (RefSeq acceptance number NM_025225.2; SEQ ID NO: 1; SEQ ID NO: 2). Mammalian orthologs of the human PNPLA3 gene include, for example, GenBank acceptance number GI:544461323 (RefSeq acceptance number XM_005567051.1, cynomolgus monkey; SEQ ID NOs. 7 and 8); GI:544461325 (RefSeq acceptance number XM_005567052.1, cynomolgus monkey; SEQ ID NOs. 11 and 12); GI:297261270 (RefSeq acceptance number XM_001109144.2, rhesus monkey) It can be found in monkey, SEQ ID NO: 9 and SEQ ID NO: 10); GI: 144226244 (RefSeq acceptance number NM_054088.3, ​​mouse; SEQ ID NO: 3 and SEQ ID NO: 4); GI: 537361027 (RefSeq acceptance number NM_001282324.1, rat; SEQ ID NO: 5 and SEQ ID NO: 6).

[0077] Further examples of PNPLA3 mRNA sequences are readily available using commonly available databases, such as GenBank, UniProt, and OMIM.

[0078] In the use of this specification, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the PNPLA3 gene, including mRNA, which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage, either in or near the portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the PNPLA3 gene. In one embodiment, the target sequence is located within the protein-coding region of PNPLA3.

[0079] 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.

[0080] In the use of this specification, the term “sequence-containing chain” refers to an oligonucleotide containing a nucleotide chain described by the sequence referred to, using standard nucleotide nomenclature.

[0081] "G," "C," "A," "T," and "U" typically represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, the terms "ribonucleotide" or "nucleotide" are also understood to refer to modified nucleotides or substituted portions, as will be further detailed below (see, for example, Table 2). Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be replaced with other portions without substantially altering the base-pairing properties of oligonucleotides containing such substituted portions. As an example not intended to be limiting, a nucleotide containing inosine as a base can base-pair with adenine, cytosine, or uracil-containing nucleotides. Thus, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the dsRNAs discussed in this invention with, for example, nucleotides containing inosine. In another embodiment, adenine and cytosine may be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form GU fluctuation base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods discussed in this invention.

[0082] 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 PNPLA3 gene in cells, such as cells in a target such as a mammalian subject.

[0083] 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 PNPLA3 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 PNPLA3 gene. Accordingly, the term "siRNA" is also used herein to refer to the RNAi as described above.

[0084] 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. Designs and tests of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, their entire contents of which are incorporated herein by reference. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein, or chemically modified in the manner described in Lima et al., (2012) Cell 150:883-894.

[0085] In another embodiment, the “iRNA” used in the compositions, uses, and methods of the present invention is double-stranded RNA and is referred herein to as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” refers to a double-stranded ribonucleic acid molecule complex having a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having “sense” and “antisense” orientations with respect to the target RNA, i.e., the PNPLA3 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) induces the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred herein to as RNA interference or RNAi.

[0086] 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 on siRNA-type molecules are encompassed in “RNAi agent” for the purposes of this specification and the claims.

[0087] 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.

[0088] 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 strands are referred to as a "hairpin loop." A hairpin loop may contain at least one unpaired nucleotide; in some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, and at least 23 or more unpaired nucleotides. 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.

[0089] If two substantially complementary strands of dsRNA are composed of another RNA molecule, these molecules may, but do not necessarily, be covalently linked. If the two strands are covalently linked between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand by means other than an uninterrupted nucleotide chain, the linking structure is called a "linker." RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs.

[0090] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a PNPLA3 target mRNA sequence, to induce cleavage of the target RNA. 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 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).

[0091] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of dsRNA extends over the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang may contain, or consist of, nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located on the 5' end, the 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0092] 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, overhanging, for example, at the 3' and / or 5' ends. In another embodiment, one or more nucleotides in the overhang are substituted with thiophosphate nucleosides. In certain embodiments, the overhang of the sense strand or the antisense strand, or both, may have an extended length greater than 10 nucleotides, for example, 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 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.

[0093] "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at that end of a double-stranded RNAi agent, i.e., there are no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded along its entire length, i.e., has no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents having a nucleotide overhang at one end (i.e., an agent with one overhang and one blunt end) or RNAi agents having nucleotide overhangs at both ends. For clarification, a "blunt-ended" dsRNA is a dsRNA with both ends blunted, i.e., has no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded along their entire length.

[0094] The terms “antisense strand” or “guide strand” refer to an iRNA strand, such as dsRNA, that includes a region substantially complementary to a target sequence, such as PNPLA3 mRNA. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as the PNPLA3 nucleotide sequence as defined herein. If the complementary region is not perfectly complementary to the target sequence, there may be mismatches in the internal or terminal regions of the molecule. Generally, the most acceptable mismatches are in terminal regions, such as within 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends of the iRNA. In one embodiment, the double-stranded RNAi agent of the present invention includes a nucleotide mismatch in the antisense strand. In another embodiment, the double-stranded RNAi agent of the present invention includes a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, within the 3' terminal nucleotide of the iRNA.

[0095] 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.

[0096] 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.

[0097] In the use of this specification, unless otherwise specified, the term “complementary” refers to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under specific conditions to form a double-stranded structure, as will be understood by those skilled in the art when used to describe a first nucleotide sequence in relation to a second nucleotide sequence. Such conditions may be stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual,” Sambrook, et al. (1989), Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically reasonable conditions that may be encountered in living organisms, may also be applicable. Those skilled in the art can determine the optimal set of conditions for the complementarity test of the two sequences, depending on the end use of the hybridized nucleotides.

[0098] For example, complementary sequences within iRNAs, such as those within dsRNAs, as described herein, include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence, spanning the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred to herein as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more mismatched base pairs, generally 5, 4, 3, or 2 or fewer, while retaining the ability to hybridize into double-stranded hybridization of up to 30 base pairs under conditions most appropriate for their end use, such as inhibition of gene expression via the RISC pathway. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, where the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, is still referred to as “perfectly complementary” for the purposes described herein.

[0099] "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.

[0100] 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.

[0101] 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 PNPLA3 gene). For example, a polynucleotide is complementary to at least a portion of PNPLA3 mRNA if its sequence is substantially complementary to an undisrupted portion of the mRNA encoding the PNPLA3 gene.

[0102] Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target PNPLA3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PNPLA3 sequence and comprise a contiguous 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.

[0103] In some embodiments, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, the antisense polynucleotide being complementary to a target PNPLA3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to an equivalent region of the nucleotide sequence of SEQ ID NO: 2, or a fragment of SEQ ID NO: 2, 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.

[0104] In another embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide, the antisense polynucleotide being complementary to a target PNPLA3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to an equivalent region of the nucleotide sequence of one of the sense strands shown in any one of Tables 3-5, 7, and 8, or one fragment of one of the sense strands shown in any one of Tables 3-5, 7, and 8, 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.

[0105] In one aspect of the present invention, the agent used in the method and composition of the present invention is a single-stranded antisense oligonucleotide molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence in the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base-pairing with the mRNA and physically interfering with the translation mechanism. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule may be about 15 to about 30 nucleotides long and have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may contain a sequence that is at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0106] In the use of this specification, “Subject” means a mammal, including primates (such as humans, non-human primates such as monkeys and chimpanzees), non-primates (such as cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, and whales), or an animal such as a bird (such as a duck or goose). In one embodiment, the subject is a human being treated or evaluated for a disease, disorder or condition that would benefit from reduced PNPLA3 gene expression and / or replication; a human being at risk of a disease, disorder or condition that would benefit from reduced PNPLA3 gene expression; a human being having a disease, disorder or condition that would benefit from reduced PNPLA3 gene expression; and / or a human being treated for a disease, disorder or condition that would benefit from reduced PNPLA3 gene expression as described herein. In one embodiment, the subject is a female human being. In another embodiment, the subject is a male human being.

[0107] In the context of this specification, the terms “to treat” or “to cure” refer to a beneficial or desired outcome, including, but not limited to, the alleviation or improvement of one or more conditions associated with PNPLA3 gene expression and / or PNPLA3 protein production, such as the presence of increased protein activity in the Hedgehog (Hh) signaling pathway, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatic inflammation, 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.

[0108] The term “reduce” in relation to the level of PNPLA3 gene expression and / or PNPLA3 protein production in the subject, or to a disease marker or symptom, means a statistically significant reduction in such levels. 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 more, but preferably to a level that is generally recognized as being within the normal range for individuals without such disease.

[0109] As used herein, “prevention” or “prevention” as used in relation to a disease, disorder, or condition that benefits from reduced expression of the PNPLA3 gene and / or production of the PNPLA3 protein means a reduced likelihood of the subject developing symptoms associated with the disease, disorder, or condition, such as, for example, the presence of increased levels of the protein in the Hedgehog signaling pathway, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fatty accumulation in the liver, inflammation of the liver, 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).

[0110] In the use of this specification, the terms “patatin-like phospholipase domain-containing PNPLA3-related disease” or “PNPLA3-related disease” refer to diseases or disorders caused by or associated with the expression of the PNPLA3 gene or the production of the PNPLA3 protein. The term “PNPLA3-related disease” includes diseases, disorders, or conditions that benefit from reduced PNPLA3 gene expression, replication, or protein activity. Non-limiting examples of PNPLA3-related diseases include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatic inflammation, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). In another embodiment, PNPLA3-related disease is non-alcoholic fatty liver disease (NAFLD). In another embodiment, PNPLA3-related disease is non-alcoholic steatohepatitis (NASH). In another embodiment, PNPLA3-related disease is cirrhosis. In another embodiment, PNPLA3-related disease is insulin resistance. In another embodiment, PNPLA3-related disease is not insulin resistance. In one embodiment, PNPLA3-related disease is obesity.

[0111] In one embodiment, the PNPLA3-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 mg 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).

[0112] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject having a PNPLA3-related disease, results in treatment of the disease (e.g., by reduction, amelioration or maintenance of an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity, and the medical history, age, weight, family history, genetic constitution of the patient being treated, the stage of the pathological process mediated by PNPLA3 gene expression, the type of prior or concomitant therapeutic agents, and other individual characteristics if any.

[0113] As used herein, a "preventive effective amount" is intended to include an amount of an iRNA agent that, when administered to a subject who has not yet experienced or presented symptoms of a PNPLA3-related disease but who may be at risk of developing the disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Amelioration of the disease can include slowing of the disease progression or a decrease in the severity of a disease that develops later. A "preventive effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the degree of disease risk, and the medical history, age, weight, family history, genetic constitution of the patient being treated, the type of prior or concomitant therapeutic agents, and other individual characteristics if any.

[0114] A "therapeutically effective amount" or "preventive effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effects at a reasonable benefit / risk ratio applicable to any therapeutic agent. The RNAi agent used in the methods of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0115] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications and that are commensurate with a reasonable benefit / risk ratio.

[0116] In the use herein, 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, talc, magnesium, calcium or zinc stearate, or steric acid), or a solvent encapsulating material involved in transporting or delivering a 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 may serve as pharmacoagulably 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) magnesium (1) Smoothing agents such as sodium lauryl sulfate and talc; (2) Excipients such as cocoa butter and suppository wax; (3) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (4) Glycols such as propylene glycol; (5) Polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (6) Esters such as ethyl oleate and ethyl laurate; (7) Agar; (8) Magnesium hydroxide Examples include buffers such as nesium and aluminum hydroxide; (15) alginic acid; (16) pyrogenic substance-free water; (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.

[0117] As used herein, the term "sample" includes an isolated collection of similar bodily fluids, cells, or tissues from a subject, as well as bodily fluids, cells, or tissues present within the subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples include samples from tissue, organs or localized regions. For example, a sample may be derived from a particular organ, a portion of an organ, or cells within a bodily fluid or these organs. In certain embodiments, the sample is from the liver (e.g., the whole liver or a particular segment of the liver or a particular type of liver cell, e.g., hepatocytes), the retina or a part of the retina (e.g., retinal pigment epithelial cells), the central nervous system or a part of the central nervous system (e.g., ventricles or choroid plexus), or the pancreas or a particular cell or part of the pancreas. In some embodiments, "a sample obtained from a subject" refers to cerebrospinal fluid collected from the subject. In a preferred embodiment, "a sample obtained from a subject" refers to blood or plasma collected from the subject. In another embodiment, "a sample obtained from a subject" refers to liver tissue (or a component thereof) or retinal tissue (or a component thereof) obtained from the subject.

[0118] II. iRNA of the present invention The present invention provides an iRNA that inhibits the expression of the PNPLA3 gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits the expression of the PNPLA3 gene in cells, such as cells in a subject, such as a mammal, including, for example, a human with a PNPLA3-related disease such as non-alcoholic fatty liver disease (NAFLD). The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during the expression of the PNPLA3 gene. The complementary region is 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 in length). Upon contact with cells expressing the PNPLA3 gene, the iRNA inhibits the expression of the PNPLA3 gene (e.g., human, primate, non-primate, or avian PNPLA3 gene) by at least approximately 10% as assayed by methods such as PCR or branched DNA (bDNA) based methods, or by protein-based methods such as immunofluorescence analysis using Western blotting or flow cytometry.

[0119] 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 PNPLA3 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.

[0120] Generally, double-stranded structures are, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 These range from 15 to 30 base pairs in length, such as 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Intermediate ranges and lengths beyond the listed ranges and lengths are also intended to be part of the present invention.

[0121] 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.

[0122] In some embodiments, the dsRNA is about 15–23 nucleotides long, or about 25–30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. It is well known in the art that dsRNA longer than about 21–23 nucleotides may serve as a substrate for Dicer. As those skilled in the art will recognize, the target region of the RNA to be cleaved is in most cases part of a larger RNA molecule, which is often an mRNA molecule. Where applicable, the “part” of the mRNA target is a continuous sequence of mRNA targets long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0123] Those skilled in the art can, for example, use approximately 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-3 3, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-1 9, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20 It will also be recognized that double-stranded regions of approximately 9 to 36 base pairs, such as ~28, 20~27, 20~26, 20~25, 20~24, 20~23, 20~22, 20~21, 21~30, 21~29, 21~28, 21~27, 21~26, 21~25, 21~24, 21~23, or 21~22 base pairs, are the main functional parts of dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs, within the range that it is processed into a functional double-strand of 15 to 30 base pairs that targets a desired RNA for cleavage, 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 PNPLA3 gene expression are not generated in the target cell by cleaving larger dsRNAs.

[0124] 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.

[0125] 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 further discussed below.

[0126] 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.

[0127] 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 is selected from the sequence group provided in any one of Tables 3-5, 7, and 8, and the antisense strand corresponding to the sense strand is selected from any one of the sequence group provided in Tables 3-5, 7, and 8. 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 PNPLA3 gene expression. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide described as the sense strand from any one of Tables 3-5, 7, and 8, and the second oligonucleotide described as the antisense strand corresponding to the sense strand from any one of Tables 3-5, 7, and 8. In one embodiment, substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0128] The sequences shown in any one of Tables 3, 4, and 7 are not listed as modified and / or conjugated sequences, but it is understood that the RNA of the iRNA of the present invention, such as the dsRNA of the present invention, may include any one of the sequences shown in any one of Tables 3-5, 7, and 8, or any one of the sequences in Tables 3-5, 7, and 8 that is modified, or any one of the sequences in Tables 3-5, 7, and 8 that is conjugated. In other words, the present invention encompasses any one of the dsRNAs in Tables 3-5, 7, and 8 that is unmodified, unconjugated, modified, and / or conjugated, as described herein.

[0129] In another embodiment, the double-stranded ribonucleic acid (dsRNA) of the present invention for inhibiting PNPLA3 expression comprises, substantially, a sense strand and an antisense strand, wherein the sense strand comprises the sense strand nucleotide sequence shown in any one of Tables 3-5, 7, and 8, and the antisense strand comprises the corresponding antisense strand nucleotide sequence shown in any one of Tables 3-5, 7, and 8.

[0130] Those skilled in the art are well aware that dsRNAs having double-stranded structures of approximately 20–23 base pairs, such as 21 base pairs, are supported as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877–6888). However, other those skilled in the art have found that shorter or longer RNA double-stranded structures can also be equally effective (Chu and Rana (2007) RNA 14:1714–1719; Kim et al. (2005) Nat Biotech 23:222–226). In the embodiments described above, due to the nature of the oligonucleotide sequences provided in any one of Tables 3–5, 7, and 8, 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 sequences having one of the sequences in Tables 3-5, 7, and 8, with only a few nucleotides missing from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotide sequences derived from one of the sequences in Tables 3-5, 7, and 8, and having the ability to inhibit PNPLA3 gene expression, differing from dsRNAs containing full-length sequences by approximately 5, 10, 15, 20, 25, or 30% or less, are intended to be within the scope of the present invention.

[0131] Furthermore, the RNAs provided in any one of Tables 3-5, 7, and 8 identify sites in the PNPLA3 transcript that are highly sensitive to RISC-mediated cleavage (see, for example, Table 9). Thus, the present invention further features iRNAs that target within one of these sequences. As used herein, an iRNA is said to target within that specific site of the RNA transcript if it promotes cleavage of the transcript somewhere within that site. Such iRNAs generally consist of about 15 consecutive nucleotides from one of the sequences provided in any one of Tables 3-5, 7, and 8, which are linked to an additional nucleotide sequence from a region adjacent to the selected sequence in the PNPLA3 gene.

[0132] Target sequences are generally about 15–30 nucleotides long, but there is a wide range of variation in the suitability of specific sequences within this range to induce cleavage of any given target RNA. The various software packages and guidelines presented herein provide guidance for identifying the optimal target sequence for any given gene target, but an empirical approach can also be taken to identify sequences within a size range that could act as the target sequence by actually or figuratively (including, for example, by computer simulation) placing a given size "window" or "mask" (21 nucleotides as an unrestricted example) on the target RNA sequence. By successively 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 for any given target size is identified. Therefore, while, for example, the sequences identified in any one of Tables 3-5, 7, and 8 represent effective target sequences, it is possible to further optimize inhibition efficiency by gradually "walking the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0133] Furthermore, it is explored that further optimization of any sequence identified in any one of Tables 3-5, 7, and 8 may be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking a window of size longer or shorter than the target RNA from that position. Again, combining this approach to creating new target candidates with testing the efficacy of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein may lead to further improvements in inhibition efficiency. Moreover, such optimized sequences may be modified to further optimize the molecule as an expression inhibitor (e.g., increased serum stability or circulating half-life, increased thermal stability, enhanced transmembrane delivery, targeting of specific sites or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes, etc.) by introducing modified nucleotides described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or considered herein.

[0134] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is not located in the center of the complementary region. When the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferable that the mismatch is limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA drug chain complementary to the PNPLA3 gene region, the RNA chain generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it may be determined whether an iRNA containing a mismatch with the target sequence is effective in inhibiting the expression of the PNPLA3 gene. Examining the effectiveness of mismatched iRNAs in inhibiting PNPLA3 gene expression is important, especially when a particular complementary region of the PNPLA3 gene is known to have polymorphic sequence variations within the population.

[0135] 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 iRNA of the present invention, where "substantially all of the nucleotides are modified" means that most are modified but not completely modified, and may include 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0136] 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, conjugate linkage, inversion linkage) or 3'-end modifications (conjugate linkage, DNA nucleotide, inversion linkage, etc.); base modifications such as substitution, base removal (debasing nucleotide), or conjugate-linking bases, for example, at stabilizing bases, at destabilizing bases, or at 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 linkages. 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.

[0137] Examples of modified RNA backbones include phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, 3'-alkylene phosphonate and chiral phosphonate including methyl and other alkyl phosphonates, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and normal 3'-5' linkages, boranophosphate having 2'-5' linked analogs thereof, and boranophosphate having reverse polarity where the adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] In another embodiment, a suitable RNA mimetic is considered for use in iRNA, in which both the sugar and nucleoside bonds, i.e., the nucleotide unit backbone, are replaced with a new group. The base units are maintained for hybridization with a suitable nucleic acid target compound. Such an oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In a PNA compound, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bind directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262, each of which is incorporated herein by reference in its entirety. Furthermore, PNA compounds suitable for use in iRNA according to the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0142] 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.

[0143] 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., alkoxy-alkoxy groups. Other exemplary modifications include 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, 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.

[0144] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the iRNA, specifically at the 3' position of the sugar on the 3' terminal nucleotide, or in the 2'-5' linked dsRNA, and at the 5' position of the 5' terminal nucleotide. The iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of the above-mentioned modified sugar structures include, specifically, U.S. Patent No. 4,981,957; U.S. Patent No. 5,118,800; U.S. Patent No. 5,319,080; U.S. Patent No. 5,359,044; U.S. Patent No. 5,393,878; U.S. Patent No. 5,446,137; U.S. Patent No. 5,466,786; U.S. Patent No. 5,514,785; U.S. Patent No. 5,519,134; U.S. Patent No. 5, U.S. Patent Nos. 567,811; U.S. Patent Nos. 5,576,427; U.S. Patent Nos. 5,591,722; U.S. Patent Nos. 5,597,909; U.S. Patent Nos. 5,610,300; U.S. Patent Nos. 5,627,053; U.S. Patent Nos. 5,639,873; U.S. Patent Nos. 5,646,265; U.S. Patent Nos. 5,658,873; U.S. Patent Nos. 5,670,633; and U.S. Patent Nos. 5,700,920, among others. The entire contents of each of the above are incorporated herein by reference.

[0145] iRNAs may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). In the use of this specification, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include deoxythymine (dT), 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; and 5-uracil (pseudouracil). Other examples of synthetic and natural nucleic acid bases include 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-daazaadenine; and 3-deazaguanine and 3-deazaadenine.Furthermore, examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, STand Lebleu, B., Ed., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed in this invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid double-strand stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276–278), making it an exemplary base substitution, especially when combined with 2'-O-methoxyethyl sugar modification.

[0146] Representative U.S. patents teaching the preparation of the specific modified nucleic acid bases and other modified nucleic acid bases described above are, as their entire contents are incorporated herein by reference, U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469. U.S. Patent Nos. 5,594,121, 5,596,091; U.S. Patent Nos. 5,614,617; U.S. Patent Nos. 5,681,941; U.S. Patent Nos. 5,750,692; U.S. Patent Nos. 6,015,886; U.S. Patent Nos. 6,147,200; U.S. Patent Nos. 6,166,197; U.S. Patent Nos. 6,222,025; U.S. Patent Nos. 6,235,887; U.S. Patent Nos. 6,380,368; U.S. Patent Nos. 6,528,640; U.S. Patent Nos. 6,639,062; U.S. Patent Nos. 6,617,438; U.S. Patent Nos. 7,045,610; U.S. Patent Nos. 7,427,672; and U.S. Patent Nos. 7,495,088.

[0147] The RNA of iRNA can 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 that includes 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 (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety, in which the ribose moiety includes an additional bridge that connects the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively “locks” the ribose 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.

[0148] 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.

[0149] 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).

[0150] Any of the aforementioned bicyclic nucleosides having one or more stereochemical sugar configurations, such as α-L-ribofuranose and β-D-ribofuranose, can be prepared (see International Publication No. 99 / 14226).

[0151] The RNA of the iRNA can also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid containing a bicyclic sugar moiety with 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”.

[0152] The iRNA of the present invention may also comprise one or more “stereoconstricted nucleotides” (“CRNs”). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable configuration, increasing its hybridization affinity to mRNA. The linker is long enough to reduce ribose ring puckering by positioning oxygen in a location optimal for stability and affinity.

[0153] 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.

[0154] Furthermore, one or more nucleotides of the iRNA of the present invention may include a hydroxymethyl-substituted nucleotide. The "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also known as an "unlocked nucleic acid" ("UNA") modification.

[0155] Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent Publication No. 8,314,227; U.S. Patent Application Publication No. 2013 / 0096289; U.S. Patent Application Publication No. 2013 / 0011922; and U.S. Patent Application Publication No. 2013 / 0313020, each incorporated herein by reference in its entirety.

[0156] 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.

[0157] Other modifications of the iRNA nucleotides 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.

[0158] A. Modified iRNA containing the motif of the present invention In certain aspects of the present invention, the double-stranded RNAi agent of the present invention includes a chemically modified agent, for example, as disclosed in U.S. Provisional Patent Application No. 61 / 561,710 filed on November 18, 2011, or PCT / US2012 / 065691 filed on November 16, 2012.

[0159] As described herein and in U.S. Provisional Patent Application No. 61 / 651,710 or PCT Application No. PCT / US2012 / 065691, excellent results can be obtained by introducing one or more motifs of three identical modifications to three consecutive nucleotides into the sense and / or antisense strands of the RNAi agent, particularly at or near the cleavage site. 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 optionally be conjugated with, for example, a GalNAc derivative ligand on the sense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0160] 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.

[0161] Accordingly, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the PNPLA3 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.

[0162] 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.

[0163] 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 also be linked, for example, by additional bases forming a hairpin, or by other non-base linkers.

[0164] 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), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT may be an overhang sequence 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.

[0165] 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.

[0166] RNAi agents contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, the single-strand 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 want to be constrained by theory, 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] If the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide bonds 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).

[0172] 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, forming 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 contains 10-30 consecutive nucleotides that are unpaired with the sense strand. 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-stranded 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.

[0173] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, 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, and 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, where 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.

[0174] In one embodiment, the sense strand of the RNAi agent includes at least one motif of three identical modifications to three consecutive nucleotides, one of which is located at a cleavage site on the sense strand.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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. The wing modifications are adjacent to the first motif or separated by at least one or more nucleotides. 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 of the first motif at or near the cleavage site, or on either side of the lead motif.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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 alterations of one or more unbound phosphate oxygens 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.

[0185] 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, particularly at the ends. The 5' end or both ends can be phosphorylated.

[0186] 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.

[0187] In one embodiment, each residue in the sense and antisense chains is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The chains may contain two or more modifications. In one embodiment, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro.

[0188] 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.

[0189] 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···”.

[0190] 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 the nucleotide in question, the alternating pattern, i.e., the alternating 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...".

[0191] 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.

[0192] 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.

[0193] 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.

[0194] In one embodiment, when three identical modification motifs of 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.

[0195] 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 on the strand. For example, the modification of the internucleotide bond may be present on all nucleotides on the sense strand and / or antisense strand; each modification of the internucleotide bond may be present in an alternating pattern on 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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 an antisense strand, independently selected from the groups 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.

[0200] 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.

[0201] 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.

[0202] 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 as follows: During the ceremony: 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 modification; 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.

[0203] In one embodiment, N a and / or N b This includes alternating pattern modifications.

[0204] 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 (e.g., it may be located at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13).

[0205] 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].

[0206] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0207] If the sense chain is represented by formula (Ic), then N bThis represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0208] If the sense chain is represented by formula (Id), then each N b Each independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N b The number of elements is 0, 1, 2, 3, 4, 5, or 6. a Each of these can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X, Y, and Z may be the same as or different from one another.

[0209] 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].

[0210] If the sense chain is represented by equation (Ia), then each N a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0211] 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 as follows: During the ceremony: 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, each sequence containing 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.

[0212] In one embodiment, N a 'and / or N b ' includes alternating pattern modifications.

[0213] In one embodiment, 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.

[0214] In one embodiment, all Y'Y'Y' motifs are 2'-OMe modified nucleotides.

[0215] 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.

[0216] 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].

[0217] If the antisense chain is expressed as equation (IIb), then N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0218] If the antisense chain is expressed as equation (IIc), then N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0219] If the antisense chain is expressed as equation (IId), then each N b' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. 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.

[0220] 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].

[0221] 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. Each of X', Y', and Z' may be the same as or different from one another. Each nucleotide in the sense and antisense strands may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands may be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0222] 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.

[0223] 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.

[0224] 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).

[0225] 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 represented by, During the ceremony: 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 ' represents an oligonucleotide sequence containing 0 to 25 modified nucleotides independently, where each sequence contains at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0 to 10 modified nucleotides independently; 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] When an RNAi agent is represented by formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a These independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0231] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a , N a ' independently represents an oligonucleotide sequence 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.

[0232] In equations (III), (IIIa), (IIIb), (IIIc), and (IIId), each of X, Y, and Z may be the same as or different from one another.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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 sense strand 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.

[0238] 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 sense strand 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.

[0239] 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.

[0240] 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.

[0241] In one embodiment, two RNAi agents, represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), are conjugated to a ligand optionally, conjugated to each other at their 5' ends and to one or both of their 3' ends. 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.

[0242] 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.

[0243] As will be described in more detail below, RNAi agents comprising a conjugate 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 conjugated to a modified subunit of the RNAi agent. For example, one or more ribonucleotide subunits of a dsRNA agent can be substituted with another moiety conjugated to a carbohydrate ligand, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit is thus substituted is referred herein to as a ribose-substituted modified subunit (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.

[0244] The ligand may be bound to a polynucleotide 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 ring atom of the cyclic carrier that binds to a selected moiety, e.g., a carbon atom or heteroatom (different from the atom that donates the skeletal linkage site). The selected moiety may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. Optionally, the selected 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.

[0245] 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.

[0246] 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 any one of Tables 3-5, 7, and 8. These agents may further contain ligands.

[0247] IV. Ligand-conjugated iRNAs Another modification of the iRNA of the present invention involves chemically linking one or more ligands, moieties, or complexes to the RNA, which enhance the activity, cell distribution, or intracellular uptake of the iRNA. Such parts include lipid portions such as the cholesterol portion (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060); thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770) and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); and thioethers such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO Aliphatic chains such as J,1991,10:1111-1118;Kabanov et al.,FEBS Lett.,1990,259:327-330;Svinarchuk et al.,Biochimie,1993,75:49-54); phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36:3651-3654;Shea et al.,Nucl.Acids Res.,1990,18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969-973; or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys.Examples include, but are not limited to, octadecylamine or the hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0248] 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.

[0249] Ligands may include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, including synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or α-helical peptides.

[0250] The ligand may also include targeting groups such as antibodies that bind to specific cell types, such as kidney cells, or cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins. The targeting groups may be thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetylglucosamine (gulucoseamine), polyhydric mannose, polyhydric fucose, glycosylated polyamino acids, polyhydric galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimetic compounds.

[0251] Other examples of ligands include dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithoglycerol Examples include lic acid, O3-(oleoyl)colenic acid, dimethoxytrityl, or phenoxazine) and peptide complexes (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole complexes, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, or AP.

[0252] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for a co-ligand; or antibodies, such as antibodies that bind to a specified cell type, such as liver cells. Ligands may also include hormones and hormone receptors. They may also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide chemical species such as polyhydric lactose, polyhydric galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyhydric mannose, or polyhydric fucose. Ligands may be lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators, for example.

[0253] 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.

[0254] 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.

[0255] The ligand-conjugate oligonucleotides of the present invention may be synthesized by using oligonucleotides having pendant-reactive functional groups, such as those derived from the addition of a binding molecule onto an oligonucleotide (described below). These reactive oligonucleotides may be reacted directly with commercially available ligands, synthesized ligands having any of the various protecting groups, or ligands having a binding site to which attachment is possible.

[0256] The oligonucleotides used in the complexes of the present invention may, conveniently and conventionally, be produced through well-known solid-phase synthesis techniques. Apparatus for such synthesis is available from several suppliers, including Applied Biosystems (Foster City, Calif.). Alternatively, any other means known in the art for such synthesis may be used. It is also known that other oligonucleotides, such as phosphorothioates and alkylated derivatives, can be prepared using similar techniques.

[0257] 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, or nucleotide or nucleoside complex precursors that already contain a binding site, or ligand-nucleotide or nucleoside complex precursors that already contain a ligand molecule, or basic units containing a non-nucleoside ligand.

[0258] When a nucleotide complex precursor already containing a binding site is used, the synthesis of a sequence-specific binding 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 binding 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.

[0259] A. Lipid complexes In one embodiment, the ligand or complex is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the complex to target tissues, such as non-renal target tissues of the body. Target tissues, for example, could be the liver, including hepatic parenchymal cells. Other molecules capable of binding to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the degradation resistance of the complex, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate the binding of serum proteins, such as HSA.

[0260] Lipid-based ligands can be used for inhibition, for example, by controlling the binding of the complex to target tissues. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the complex to the kidneys.

[0261] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the complex is distributed to non-renal tissues. However, the affinity is preferably not so strong as to prevent the HSA ligand binding from being reversed.

[0262] In another preferred embodiment, the lipid-based ligand binds weakly to or does not bind at all to the HSA so that the complex is preferably distributed to the kidney. Other portions that target renal cells may also be used instead of, or in addition to, the lipid-based ligand.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] Peptides or peptide mimetic drugs may be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, a bound peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane-transfer sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). RFGF analogues containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 14)) 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. Sequences from, for example, HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 15)) and Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 16)) have been shown to function as delivery peptides. Peptides or peptide mimetic drugs can be encoded by random sequences of DNA, such as peptides identified from phage-display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). For cell targeting purposes, examples of peptides or peptide mimetic drugs anchored to dsRNA activators via incorporated monomer units include arginine-glycine-aspartate (RGD) peptides or RGD mimetic drugs. The peptide moiety can range in length from approximately 5 to 40 amino acids. The peptide moiety may have structural modifications that increase stability or induce conformational properties. Any of the structural modifications described below may be used.

[0267] 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.

[0268] "Cell-permeable peptides" can penetrate cells such as microbial cells, including bacterial or fungal cells, or mammalian cells, including human cells. Microbial cell-permeable peptides may be, for example, α-helical linear peptides (e.g., LL-37 or ceropin P1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidine). Cell-permeable peptides may also contain nuclear localization signals (NLS). For example, cell-permeable peptides may be bifidopphimotic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0269] C. Carbohydrate Conjugate 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. Specific monosaccharides include sugars with a HBV or higher (e.g., HBV, C6, C7, or C8); disaccharides include sugars having two or three monosaccharide units (e.g., HBV, C6, C7, or C8).

[0270] In one embodiment, the carbohydrate complex used in the composition and method of the present invention is a monosaccharide. In one embodiment, the monosaccharide is [ka] These include N-acetylgalactosamine.

[0271] In another embodiment, the carbohydrate complex used in the compositions and methods of the present invention is [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0272] Another representative carbohydrate complex used in the embodiments described herein is: [ka] (In the formula, Examples include, but are not limited to, a (where either X or Y is an oligonucleotide and the other is hydrogen) array.

[0273] 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.

[0274] Further carbohydrate complexes 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.

[0275] D. Linker In some embodiments, the complexes or ligands described herein may be attached to the iRNA oligonucleotide by various linkers, which may be cleavable or incleavable.

[0276] 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, 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, alkylherocyclylalkynyl, alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkyl, alkynyl heterocyclylalkenyl, alkynylherocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenyl heteroaryl, alkynylhereroaryl, and other atomic chains, one or more of which 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 16 atoms, or 8 to 16 atoms.

[0277] The cleavable linking group is sufficiently stable outside the cell but is cleaved upon entry into the target cell, releasing the two parts held together by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more, or at least about 100 times faster in the target cell or under a first standard condition (which may be selected to mimic or be equivalent to intracellular conditions) than in the target blood or under a second standard condition (which may be selected to mimic or be equivalent to conditions found in blood or serum).

[0278] 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.

[0279] 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.

[0280] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into a linker may depend on the target cell. For example, a ligand targeting the liver may link to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and therefore linkers are cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0281] Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synovial cells.

[0282] Generally, the suitability of candidate cleavable linkers can be evaluated by testing the ability of a degrading agent (condition) to cleave the candidate linker. It is also desirable to test the candidate cleavable linker's resistance to cleavage in the blood or in contact with other non-target tissues. Therefore, the relative susceptibility to cleavage between a first and second condition can be determined, with the first condition selected to demonstrate cleavage in target cells and the second condition selected to demonstrate cleavage in other tissues or in biological fluids such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or in whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and then confirm it with further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0283] i. Redox-cleavable linking groups In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). Methods described herein can be relied upon to determine whether a candidate cleavable linking group is a suitable “reductively cleavable linking group” or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent. For example, a candidate may be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. One candidate compound is cleaved by up to about 10% in blood. In other embodiments, useful candidate compounds are degraded at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using a standard enzyme kinetics assay under conditions selected to mimic an extracellular medium, compared to conditions selected to mimic an extracellular medium.

[0284] ii. Phosphate-based cleavable linking groups In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group can be cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves the phosphate group in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0285] iii. Acid-cleavable linking group In another embodiment, the cleavable linker includes an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, or less) or by an active agent such as an enzyme that can act as a general acid. Within cells, certain low-pH organelles such as endosomes and lysosomes can provide an environment for cleaving acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid-cleavable linking group may have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when the carbon is attached to the oxygen of the ester (alkoxy group), it is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0286] 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.

[0287] 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.

[0288] In one embodiment, the iRNA of the present invention conjugates with a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrates conjugated with the linker of the composition and method of the present invention include: [ka] [ka] [ka] (In the formula, Examples include, but are not limited to, a (where either X or Y is an oligonucleotide and the other is hydrogen) array.

[0289] 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.

[0290] In one embodiment, the dsRNA of the present invention is Formulas (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 be interrupted or terminated by one or more of these; R 2A , R2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each occurrence is independently: absence, 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 conjugates 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 Formula (XXXV), [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).

[0291] Suitable divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above as formulas II, VII, XI, X, and XIII.

[0292] 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.

[0293] 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.

[0294] In the context of this invention, a "chimeric" iRNA compound or "chimeras" is an iRNA compound, preferably a dsRNA, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to give the iRNA increased resistance to nuclease degradation, increased intracellular uptake, and / or increased binding affinity to a target nucleic acid. The additional region of the iRNA may act as an enzyme substrate capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded molecule. Therefore, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region, comparable results are often obtained with shorter iRNAs. Cleavage of RNA targets can conventionally be detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0295] In some cases, the RNA of iRNA can be modified with non-ligand groups. Several non-ligand molecules are conjugated to iRNA to enhance its activity, cell distribution, or intracellular uptake, and procedures for performing such conjugations are available in the academic literature.These non-ligand portions include lipid portions such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), and thiocholesterol (Oberhauser et al., Nucl. Acids Aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & It contains Nucleotides (1995, 14:969), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).Representative U.S. patents teaching the preparation of such RNA complexes are listed above. A typical conjugate binding protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group is then reacted with a molecule to which it will be conjugate using an appropriate coupling or activating reagent. The conjugate binding 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.

[0296] V. Delivery of iRNA by invention For example, delivery of the iRNA of the present invention to cells, such as cells within a subject (e.g., a subject that requires it, such as a human subject with a disease, disorder, or condition related to PNPLA3 gene expression), 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 target. 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.

[0297] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNA of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication No. 94 / 02595, whose entire contents are incorporated herein by reference). For in vivo delivery, factors to be considered for delivering the iRNA molecule include, for example, the biological stability of the delivery molecule, prevention of nonspecific effects, and accumulation of the delivery molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or transplantation into tissue or local administration of the formulation. Local administration to the treatment site maximizes the local concentration of the active substance, limits exposure of systemic tissues to the active substance which could otherwise be harmed or degraded by the active substance, and allows for administration of lower total doses of the iRNA molecule. Several studies have shown successful gene product knockdown when iRNA is administered locally. For example, intravitreal injection of VEGF dsRNA in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ., et al (2003) Mol.Vis.9:210-216) both demonstrated the prevention of neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol.Ther.11:267-274) and extended the survival time of mice with tumors (Kim, WJ., et al (2006) Mol.Ther.14:343-350; Li, S., et al (2007) Mol.Ther.15:515-523).RNA interference can be administered to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602), and to the lungs by intranasal administration (Howard, KA., et al. (2006) Mol.Ther.14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med.11:50-55) Successful local delivery has been demonstrated. To treat diseases, or to administer iRNA systemically, RNA can be modified or, alternatively, delivered using drug delivery systems; both methods act to prevent rapid degradation of dsRNA by endogenous and exonucleases. Modification of RNA or pharmaceutical carriers can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical bonding of lipophilic groups such as cholesterol to enhance intracellular uptake and prevent degradation. For example, when an iRNA that counteracts ApoB, conjugated to the lipophilic cholesterol portion, was systemically injected into mice, apoB mRNA knockdown was induced in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of iRNA to aptamers has been shown to suppress tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015).In alternative embodiments, iRNA may be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of the iRNA molecule (which is negatively charged) and also enhance interactions with the negatively charged cell membrane, enabling efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may be induced to bind to iRNA or form vesicles or micelles that enclose the iRNA (see, e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of iRNA when administered systemically. Methods for preparing and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), cited above; Verma, UN., et al (2003), cited above), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441:111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME., et al. Examples include al (2008) Pharm. Res., published online on August 16; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, for systemic administration, the iRNA forms a complex with cyclodextrin. Methods of administering iRNA and cyclodextrin and pharmaceutical compositions are described in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

[0298] A. Vector encoding the iRNA of the present invention PNPLA3 gene-targeting iRNAs can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International Publication No. 00 / 22113; Conrad, International Publication No. 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (from a few hours to several weeks) or persistent (from several weeks to several months or more), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which may be embedded or non-embedded vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (see Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0299] 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.

[0300] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for iRNA expression described herein can be generated using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial suppliers. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors may include systemic administration, such as intravenous or intramuscular administration; administration to target cells explanted from a patient and subsequent reintroduction into the patient; or any other means that allows introduction into desired target cells.

[0301] iRNA expression plasmids can be translocated into target cells as complexes with cationic lipid carriers (e.g., oligofectamines) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid translocation for iRNA-mediated knockdown, targeting different regions of the target RNA over a period of more than one week, is also explored in this invention. Successful introduction of the vector into host cells can be monitored using various known methods. For example, transient translocation can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable translocation into cells in vitro can be ensured by using markers that provide the translocated cells with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0302] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentivirus vectors and Moloney's mouse leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, including vaccinia virus vectors, or avipox, including canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not be incorporated into the cell genome. The construct may optionally include a viral sequence for translocation. Alternatively, the construct may be incorporated into an episomal replication vector, such as EPV and EBV vectors. Constructs for the recombinant expression of iRNAs generally require regulatory factors, such as promoters and enhancers, to ensure iRNA expression in target cells. Other aspects of vectors and constructs that are considered are described in more detail below.

[0303] A vector useful for delivering iRNA contains sufficient regulatory factors (promoters, enhancers, etc.) to express the iRNA in the desired target cells or tissues. These regulatory factors can be selected to provide either constitutive or regulatory / inducible expression.

[0304] iRNA expression can be precisely regulated using inductive regulatory sequences sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J.8:20-24). Suitable inductive expression systems for regulating dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, dimerizing chemical inducers, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0305] Viral vectors containing nucleic acid sequences encoding iRNA can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and its integration into host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors to facilitate delivery of the nucleic acid to the patient. For more details on retroviral vectors, see Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to generate stem cells that exhibit higher resistance to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors that may be considered include, for example, HIV-based vectors described in U.S. Patent No. 6,143,520; U.S. Patent No. 5,665,557; and U.S. Patent No. 5,981,276, which are incorporated herein by reference.

[0306] Adenoviruses are also being considered for use in iRNA delivery according to the present invention. Adenoviruses are particularly attractive vehicles for delivering genes, for example, to the airway epithelium. Adenoviruses infect the airway epithelium naturally, causing mild disease. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapies. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors for transferring genes into the airway epithelium of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); International Publication No. 94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing the iRNA addressed in this invention, a method for constructing a recombinant AV vector, and a method for delivering the vector to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0307] Adeno-associated virus (AAV) vectors can also be used to deliver the iRNA of the present invention (Walsh et al., Proc.Soc.Exp.Biol.Med.204:289-300(1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNA may be expressed as two distinct complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNAs addressed in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described herein by reference in their entirety: Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Publication No. 94 / 13788; and International Publication No. 93 / 24641.

[0308] Other viral vectors suitable for delivering the iRNA of the present invention include, for example, vaccinia viruses such as modified virus Ankara (MVA) or attenuated vaccinia such as NYVAC, and poxviruses such as avipox such as fowlpox or canarypox.

[0309] The affinity of a viral vector can be modified, if necessary, by pseudotyping the vector with coat proteins or other surface antigens from other viruses, or by substitution with capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mocola, etc. AAV vectors can be genetically engineered to target different cells by expressing different capsid protein serotypes; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, whose entire disclosure is incorporated herein by reference.

[0310] A vector-based drug may contain a vector in an acceptable diluent, or a sustained-release matrix in which a gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be generated intact from recombinant cells, the drug may contain one or more cells that generate the gene delivery system.

[0311] VI. Pharmaceutical composition of the 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 iRNA-containing pharmaceutical composition is useful for treating diseases or disorders related to the expression or activity of the PNPLA3 gene.

[0312] Such pharmaceutical compositions are formulated based on the delivery method. One example is a composition formulated for systemic administration via parenteral delivery, for example, by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma, for example, by intracerebral infusion, such as by continuous pump infusion. The pharmaceutical compositions of the present invention may be administered in doses sufficient to inhibit the expression of the PNPLA3 gene.

[0313] The pharmaceutical composition of the present invention may be administered in a dose sufficient to inhibit the expression of the PNPLA3 gene. Generally, appropriate doses of the iRNA of the present invention range from about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient per day, generally ranging from about 1 to 50 mg per kilogram of body weight per day. For example, dsRNA may be administered in single doses of about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg. Repeated-dose regimens may include regular administrations of therapeutic doses of iRNA, for example, every other day or annually. In certain embodiments, iRNA is administered about once a month to about once every three months (i.e., about once every three months). After the initial treatment regimen, medications can be administered at a lower frequency.

[0314] 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. Estimation of the effective dose and in vivo half-life of individual iRNAs incorporated in this invention can be carried out using conventional procedures, as known in the art, or based on in vivo studies using appropriate animal models.

[0315] Advances in mouse genetics have led to the creation of several mouse models for the study of various human diseases, including disorders that benefit from reduced PNPLA3 expression. Such models can be used for in vivo testing of drugs and for determining effective therapeutic doses. Suitable dietary and genetic mouse models are discussed in Kanuri and Bergheim (Int.J.Mol.Sci.(2013)14:11963-11980).

[0316] The pharmaceutical compositions of the present invention may be administered in several ways, depending on whether topical or systemic treatment is desired and on the treatment area. Administration may be topical (e.g., by a transdermal patch), transpulmonary by inhalation or blowing of powder or fume, including by a nebulizer; intratracheal, intranasal, transepidermal and transdermal, oral or parenteral. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal administration via an implantable device, for example; or intracranial administration, such as within the brain parenchyma, subarachnoid space or ventricles.

[0317] iRNAs can be delivered in a manner that targets specific tissues, such as the liver (e.g., parenchymal cells of the liver).

[0318] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., may be necessary or desirable. Covered condoms, gloves, etc., may also be useful. Suitable topical formulations include those in which the iRNA addressed in this invention is in a miscible material with topically delivered substances such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), 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.

[0319] A. iRNA preparations containing membrane-like molecular assemblies The iRNAs used in the compositions and methods of the present invention may be formulated for delivery within membrane-like molecular assemblies, such as liposomes or micelles. In this specification, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one or more bilayers. Liposomes include monolayer or multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition but may optionally. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to a biological membrane, when liposomes are applied to tissue, the liposomal bilayer fuses with the cell membrane bilayer. As the fusion of the liposome and cell progresses, the internal aqueous contents containing the iRNA are delivered into the cell, where the iRNA can specifically bind to target RNA and mediate its delivery. In some cases, liposomes are also specifically targeted, for example, to induce iRNAs into specific cell types.

[0320] Liposomes containing iRNA agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed without the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid complex. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid, and lauroyl sarcosine. Next, the iRNA agent preparation is added to the micelles containing the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain the iRNA agent liposome preparation.

[0321] If necessary, a support compound, for example, that aids 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). pH may also be adjusted to aid condensation.

[0322] A method for generating a stable polynucleotide delivery vehicle by incorporating a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle is further described, for example, in International Publication No. 96 / 37194, which is incorporated herein by reference in its entirety. Liposome formation is described by Felgner, PLet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. al.Biochim.Biophys.Acta 557:9,1979;Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978;Mayhew,et al.Biochim.Biophys.Acta 775:169,1984;Kim,et al.Biochim.Biophys.Acta 728:339,1983; and Fukunaga, et al. This may also include one or more embodiments of the exemplary methods described in al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing appropriately sized lipid aggregates for use as delivery vehicles include sonication and combinations of freeze-thaw and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). If consistently small (50–200 nm) and relatively uniform aggregates are desired, micro-solution preparation may be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable to packing iRNA drug preparations into liposomes.

[0323] Liposomes are broadly classified into two classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are taken into the endosome. Due to the acidic pH inside the endosome, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0324] pH-sensitive or negatively charged liposomes do not form complexes with nucleic acids; rather, they encapsulate them. Because nucleic acids and lipids both have similar charges, repulsion occurs rather than complex formation. Nevertheless, some nucleic acids are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used in culture to deliver nucleic acids encoding thymidine kinase genes to cell monolayers. Expression of exogenous genes was detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0325] One major type of liposome composition contains phospholipids in addition to naturally derived phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusion liposomes are mainly formed from dioleoyl sphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC. Yet another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0326] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; U.S. Patent Nos. 5,171,678; International Publication No. 94 / 00569; International Publication No. 93 / 24640; International Publication No. 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0327] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied, and their efficacy in drug delivery to the skin has been evaluated. Cyclosporine A was delivered into the dermis of mouse skin using nonionic liposome formulations containing Novasome® I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome® II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether). The results suggested that such nonionic liposome systems are effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. STPPharma.Sci., 1994, 4(6)466).

[0328] Liposomes also include “stereostabilized” liposomes, which, as used herein, refer to liposomes containing one or more specialized lipids, which, when incorporated into liposomes, result in an improved cyclic lifespan compared to liposomes lacking such specialized lipids. 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., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0329] 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). Both 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.).

[0330] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes cannot efficiently fuse with the plasma membrane, but they can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0331] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; they can encapsulate a wide range of water- and lipid-soluble drugs; and they can protect iRNA agents encapsulated within their internal compartments from metabolism and degradation (Rosoff, “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Key considerations in the preparation of liposomal formulations include the lipid surface charge, vesicle size, and aqueous capacity of the liposomes.

[0332] Using the positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), small liposomes can be formed, which spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with negatively charged lipids in the cell membrane of tissue culture cells, resulting in iRNA delivery (for a description of DOTMA and its use in combination with DNA, see, for example, Felgner, Plet al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355).

[0333] The ADOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin® (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to cultured tissue cells, and it contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complex thus prepared spontaneously adheres to negatively charged cell surfaces, fuses with the plasma membrane, and efficiently delivers functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl portion is linked by an ester rather than an ether linkage.

[0334] Other reported cationic lipid compounds include those conjugated with a variety of moieties, such as carboxyspermine, which conjugates with 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).

[0335] Another cationic lipid complex involves lipid derivatization by cholesterol ("DC-Chol") combined with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine with DOPE, has been reported to be effective for translocation in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient translocation than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California), and lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for oligonucleotide delivery are described in International Publication No. 98 / 39359 and International Publication No. 96 / 37194.

[0336] Liposome formulations are particularly well-suited for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver iRNA agents into the skin. In some implementations, liposomes are used to deliver iRNA agents to epidermal cells and to enhance the penetration of iRNA agents into skin tissues, such as within the skin. For example, liposomes can be applied topically. Topical delivery of therapeutic drugs formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol.2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. Jand Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. Rand Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987.

[0337] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied, and their efficacy in drug delivery to the skin has been determined. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0338] iRNA-containing liposomes can be highly deformable. Such deformability allows the liposomes to penetrate pores smaller than the average radius of the liposome. Transferosomes, for example, are a type of deformable liposome. Transferosomes can be created by adding surface edge activators, usually surfactants, to a standard liposome composition. Transferosomes containing iRNA agents can be delivered subcutaneously, for example by infection, to deliver the iRNA agent to keratinocytes in the skin. To cross intact mammalian skin, the lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of a suitable transcutaneous gradient. Furthermore, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and often self-load.

[0339] Other formulations to which the present invention can be applied are described in U.S. Provisional Patent Application No. 61 / 018,616, filed on 2 January 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed on 2 January 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed on 26 March 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed on 22 April 2008; and U.S. Provisional Patent Application No. 61 / 051,528, filed on 8 May 2008. PCT Application PCT / US2007 / 080331, filed on 3 October 2007, also describes formulations to which the present invention can be applied.

[0340] Transfersomes are yet another type of liposome, highly deformable lipid aggregates, that are attractive candidates for drug delivery vehicles. Because they are so highly deformable, transfersomes can be described as lipid droplets that can easily penetrate through pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to skin pore shapes), self-repair, and often reach and self-load their targets without fragmentation. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a serum albumin-containing solution.

[0341] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and grading the properties of the many different types of surfactants, both natural and synthetic, is the use of the hydrophile / lipophile balance (HLB). The properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying different surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0342] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used across a wide pH range. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Examples of nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, also belong to this class. Polyoxyethylene surfactants are the most commonly found components of the nonionic surfactant class.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] The use of surfactants in pharmaceuticals, formulations, and emulsions is outlined (Rieger, “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, NY, 1988, p.285).

[0347] 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.

[0348] 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 vigorous mixing is preferable to provide smaller micelles.

[0349] 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.

[0350] 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.

[0351] To deliver a micelle formulation as a spray, the formulation can be placed in a fumigant metering and dispensing device, and the spray can be loaded into the device. Under pressurization, the spray is in liquid form within the metering and dispensing device. The ratio of components is adjusted so that the aqueous phase and the spray phase are one, i.e., a single phase. If there are two phases, the metering and dispensing device needs to be shaken, for example, before dispersing a portion of the contents through a metering valve. The dispensing dose of the pharmaceutical is sprayed in a fine mist from the metering valve.

[0352] 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.

[0353] 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.

[0354] B. Lipid particles For example, iRNAs such as the dsRNA of the present invention may be completely encapsulated in a lipid formulation such as LNPs or other nucleic acid-lipid particles. The terms “lipid nanoparticles” or “LNP” refer to vesicles containing a lipid layer that encapsulate a nucleic acid molecule, such as iRNA or a plasmid on which iRNA is transcribed, which is a pharmaceutically active molecule. LNPs are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601 and 8,058,069, the entire contents of which are incorporated herein by reference.

[0355] 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 encapsulating condensant-nucleic acid complexes as described in International Publication No. 00 / 03683. The particles of the present invention are substantially non-toxic, typically having an average diameter of about 50 nm to 150 nm, more typically about 60 nm to 130 nm, more typically about 70 nm to 110 nm, and most typically about 70 nm to 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.

[0356] In one embodiment, the ratio (mass / mass ratio) of lipids to drugs (e.g., lipid to dsRNA ratio) is in the range of approximately 1:1 to approximately 50:1, approximately 1:1 to approximately 25:1, approximately 3:1 to approximately 15:1, approximately 4:1 to approximately 10:1, approximately 5:1 to approximately 9:1, or approximately 6:1 to approximately 9:1. Ranges between the ranges cited above are also considered to be part of the present invention.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] Conjugate-bound lipids that inhibit particle aggregation may be polyethylene glycol (PEG)-lipids, including, without limitation, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA complexes may be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C8). The amount of conjugate-bound lipids that inhibit particle aggregation may be 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0362] 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.

[0363] In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be prepared using the lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, whose contents are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Each stock solution in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be combined in a molar ratio, for example, 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (for example, in sodium acetate at pH 5) so that the final ethanol concentration is about 35–45% and the final sodium acetate concentration is about 100–300 mM. Lipid-dsRNA nanoparticles typically form spontaneously during mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, such as a Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be replaced with phosphate-buffered saline (PBS) at approximately pH 7, such as approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. [ka]

[0364] The LNP01 formulation is described, for example, in International Publication No. 2008 / 042973, which is incorporated herein by reference.

[0365] Additional exemplary lipid dsRNA preparations are listed in Table 1.

[0366] [Table 1]

[0367] [Table 2]

[0368] [Table 3]

[0369] 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.

[0370] Formulations containing XTC are described, for example, in U.S. Provisional Application No. 61 / 148,366 filed on 29 January 2009; U.S. Provisional Application No. 61 / 156,851 filed on 2 March 2009; U.S. Provisional Application No. 1 filed on 10 June 2009; U.S. Provisional Application No. 61 / 228,373 filed on 24 July 2009; U.S. Provisional Application No. 61 / 239,686 filed on 3 September 2009; and International Application PCT / US2010 / 022614 filed on 29 January 2010.

[0371] 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.

[0372] Formulations containing ALNY-100 are described, for example, in the international application PCT / US 09 / 63933, filed on November 10, 2009, which is incorporated herein by reference.

[0373] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed on 5 May 2009, and International Application PCT / US10 / 33777, filed on 5 May 2010, which are incorporated herein by reference.

[0374] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, suspensions or solutions in water or aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. In some embodiments, the oral formulation is administered in combination with one or more osmotic surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, combinations of osmotic enhancers are used, such as fatty acid / salt combined with bile acid / salt. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA addressed in this invention may be delivered orally in granular form, including spray-dried particles, or may be complexed to form micro or nanoparticles.Examples of DsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pullulans, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexyl Examples include acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, methyl polyacrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-coglycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparations are described in detail in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Patent No. 6,747,014, respectively, which are incorporated herein by reference.

[0375] Compositions and formulations for parenteral, intracerebral (intracerebral), subarachnoid, intraventricular, or intrahepatic administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including but not limited to osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0376] 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.

[0377] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage forms, can be prepared according to the prior art well known in the pharmaceutical industry. Such art involves the step of combining the active ingredient with a pharmaceutical carrier or excipient. Generally, formulations are prepared by uniformly and closely combining the active ingredient with a liquid carrier or an ultrafine particle solid carrier or both, and then shaping the product if necessary.

[0378] 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.

[0379] C. Additional formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid, usually in the form of droplets with a diameter greater than 0.1 μm (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and See Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p.335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301. Emulsions are often biphasic systems containing two immiscible liquid phases that are closely mixed and dispersed from one another. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed in a bulk oily phase as microdroplets, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed in a bulk aqueous phase as microdroplets, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to a dispersed phase and an active agent, which may exist as a solution in either an aqueous or oily phase, or as a separate phase itself. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions can also be multi-phase emulsions, such as oil-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer specific advantages that simple two-component emulsions do not. Among these, a multi-phase emulsion in which individual oil droplets of an o / w emulsion surround smaller water droplets constitutes a w / o / w emulsion. Similarly, an oil droplet system encapsulated in small water spheres and stabilized within a continuous oily phase provides an o / w / o emulsion.

[0380] Emulsions are characterized by having little to no thermodynamic stability. Often, the dispersed or discontinuous phases of an emulsion are well dispersed externally or within the continuous phase and maintained in this form through emulsifiers or means of increasing the formulation viscosity. In the case of emulsion-type ointment bases and creams, any of the emulsion phases may be semi-solid or solid. Another means of stabilizing an emulsion involves the use of emulsifiers, which may be incorporated into any of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorbent bases, and finely dispersed solids (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0381] Synthetic surfactants, also known as surfactants, have a wide range of applications in emulsion formulations and are outlined in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p.199). Surfactants are typically amphiphilic, containing both hydrophilic and hydrophobic moieties. The ratio of hydrophilic to hydrophobic groups is called the hydrophilic / lipophilic balance (HLB) of a surfactant and is a useful means of classifying and selecting surfactants in the preparation of pharmaceutical formulations. Surfactants can be classified into different classes based on the properties of their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0382] 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.

[0383] 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).

[0384] 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.

[0385] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can readily support microbial growth; therefore, preservatives are frequently incorporated into these formulations. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may include free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene; reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0386] 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.

[0387] ii. Microemulsion In one embodiment of the present invention, the iRNA and nucleic acid composition is prepared as a microemulsion. A microemulsion can be defined as a single optically isotropic and thermodynamically stable solution of water, oil, and an amphiphilic substance (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, a microemulsion is a system prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, which is generally an alcohol of intermediate chain length, to form a clear system. Therefore, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surfactant molecules (Leung and Shah, Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are typically prepared through a combination of 3 to 5 components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used, as well as the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.271).

[0388] 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.

[0389] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with co-surfactants. Typically, co-surfactants, which are short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant coating, resulting in an irregular coating due to gaps between surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may, but is not limited to, water, aqueous solutions of pharmaceuticals, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0390] 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.

[0391] The microemulsion of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and enhance the absorption of the iRNA and nucleic acids of the present invention. Penetration enhancers used in the microemulsion of the present invention can be classified into one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0392] iii. Particulates The iRNA agent of the present invention may be incorporated into particles, such as microparticles. Microparticles can be produced by spray drying, but they may also be produced by other methods, including freeze-drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0393] iv. Penetration enhancers In one embodiment, the present invention provides efficient delivery of nucleic acids, particularly iRNAs, to animal skin using various penetration enhancers. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been found that even non-lipophilic drugs can cross cell membranes if the membrane being traversed is treated with a penetration enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0394] 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.

[0395] 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).

[0396] 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).

[0397] The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38; Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as osmotic enhancers. Therefore, the term “bile salt” includes any of the natural components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucoseic acid (sodium glucose), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE).(See, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0398] Chelating agents used in connection with the present invention can be defined as compounds that remove metal ions from solution by forming complexes with them, thereby improving iRNA absorption through mucous membranes. With regard to their use as penetration enhancers in the present invention, since most DNA nucleases require divalent metal ions for catalytic activity and are inhibited by chelating agents, chelating substances have the additional advantage of also acting as deoxyribonuclease inhibitors (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating substances include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamine). (See, for example, Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0399] 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).

[0400] Substances that enhance iRNA uptake at the cellular level can also be added to the pharmaceuticals and other compositions of the present invention. For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188, granted to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (International Publication No. 97 / 30731, granted to Lollo et al.) are also known to enhance dsRNA uptake within cells.Examples of commercially available trait transfer reagents include, for example, Lipofectamine(trademark) (Invitrogen; Carlsbad, CA), Lipofectamine 2000(trademark) (Invitrogen; Carlsbad, CA), 293fectin(trademark) (Invitrogen; Carlsbad, CA), Cellfectin(trademark) (Invitrogen; Carlsbad, CA), DMRIE-C(trademark) (Invitrogen; Carlsbad, CA), FreeStyle(trademark)MAX(Invitrogen; Carlsbad, CA), and Lipofectamine(trademark) 2000. CD (Invitrogen; Carlsbad, CA), Lipofectamine(TM) (Invitrogen; Carlsbad, CA), iRNAMAX(Invitrogen; Carlsbad, CA), Oligofectamine(TM) (Invitrogen; Carlsbad, CA), Optifect(TM) (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent(Roche;Grenzacherstrasse,Switzerland), DOTAP Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), DOSPER Liposomal Transfection Reagent(Grenzacherstrasse,Switzerland), or Fugene(Grenzacherstrasse,Switzerland), Transfectam(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).

[0401] Glycols such as ethylene glycol and propylene glycol; pyrroles such as 2-pyrrole; azone; and other active ingredients including terpenes such as limonene and menthone can be used to enhance the penetration of administered nucleic acids.

[0402] v. Carrier Certain compositions of the present invention also incorporate a carrier compound during formulation. In the use herein, “carrier compound” or “carrier” may refer to a nucleic acid or analogue that is inactive (i.e., not biologically active itself) but is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids, for example, by degrading biologically active nucleic acids or facilitating their removal from circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter, may result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory storage sites, possibly due to competition between the carrier compound and nucleic acid for the normal receptor. For example, the recovery of partial phosphorothioate dsRNAs in liver tissue may be reduced when administered concurrently with polyinosinate, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0403] 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).

[0404] 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.

[0405] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil bases. The solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients that do not cause adverse nucleic acid reactions and are suitable for oral administration may be used.

[0406] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0407] vii. Other ingredients The compositions of the present invention may further contain other auxiliary components found in conventional pharmaceutical compositions, at levels of use established in the art. Therefore, for example, the compositions may contain additional suitable pharmacologically active materials such as antipruritics, tannins, topical anesthetics, or anti-inflammatory agents, or additional materials useful for physically compounding the various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not excessively interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0408] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0409] In some embodiments, the pharmaceutical compositions discussed herein comprise (a) one or more iRNA compounds, and (b) one or more agents that function by a non-iRNA mechanism and are useful for treating hemolytic disorders. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-lipid agents, antiviral agents, and / or anti-fibrotic agents.

[0410] In addition, other substances commonly used to protect the liver, such as silymarin, may also be used in combination with the iRNAs described herein. Other agents useful for treating liver disease include terbivudine, entecavir, telaprevir, and protease inhibitors such as those disclosed in U.S. Patent Application Publication 2005 / 0148548, U.S. Patent Application Publication 2004 / 0167116, and U.S. Patent Application Publication 2003 / 0144217 granted to Tung et al., and U.S. Patent Application Publication 2004 / 0127488 granted to Hale et al.

[0411] The toxicity and therapeutic effects of such compounds can be determined by standard pharmaceutical procedures, for example, in cell cultures or experimental animals to determine the LD50 (lethal dose in 50% of the population) and ED50 (therapeutably effective dose in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a high therapeutic index are preferred.

[0412] Data obtained from cell culture assays and animal experiments can be used to formulate dosage ranges for human use. Doses of the compositions discussed in this invention are generally within the range of circulating concentrations, including the ED50, which is minimally or completely toxic. Doses may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods discussed in this invention, the therapeutically effective dose can first be estimated from a cell culture assay. Doses may be formulated in animal models to achieve the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence, including the IC50 (i.e., the test compound concentration that achieves maximum half-dose inhibition of symptoms) determined in cell culture (e.g., achieving a reduction in polypeptide concentration). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0413] The iRNAs discussed herein may be administered in combination with other known agents effective in treating pathological processes mediated by PNPLA3 expression, in addition to the administrations considered above. In any case, the physician administering the treatment may adjust the amount and timing of iRNA administration based on the results observed using standard means of efficacy known in the art or described herein.

[0414] VII. Methods to inhibit PNPLA3 expression The present invention also provides a method for inhibiting the expression of the PNPLA3 gene in cells. This method includes the step of contacting cells with an RNAi agent, such as a double-stranded RNAi agent, in an amount effective to inhibit the expression of PNPLA3 in cells, thereby inhibiting the expression of PNPLA3 in cells.

[0415] For example, contact between an RNAi agent, such as a double-stranded RNAi agent, and cells may be carried out either in vitro or in vivo. Contact between an RNAi agent and cells in vivo includes, for example, bringing cells or cell populations within a subject, such as a human subject, into contact with the RNAi agent. A combination of in vitro and in vivo methods for cell contact is also possible. Cell contact may be direct or indirect, as described above. Furthermore, cell contact may also be achieved by a targeted ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeted ligand is, for example, a carbohydrate moiety such as GalNAc3 ligand, or any other ligand that induces the RNAi agent to a site of interest.

[0416] In one embodiment, contact between iRNA and cells includes “introduction” or “delivery of iRNA into cells” by promoting or carrying out uptake or absorption into cells. Absorption or uptake of iRNA can be carried out by spontaneous diffusion or activated cell processes, or by auxiliaries or devices. Introduction of iRNA into cells may be in vitro and / or in vivo. For example, in the case of in vivo introduction, iRNA can 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 and / or are known in the art.

[0417] The term “inhibit” as used herein is interchangeable with “reduce,” “silence,” “downcontrol,” “suppress,” and other similar terms, and includes all levels of inhibition.

[0418] The phrase "inhibit PNPLA3 expression" refers to the inhibition of expression of any PNPLA3 gene (e.g., mouse PNPLA3 gene, rat PNPLA3 gene, monkey PNPLA3 gene, or human PNPLA3 gene) as well as variants or mutants of the PNPLA3 gene. Therefore, the PNPLA3 gene may be a wild-type PNPLA3 gene, a mutant PNPLA3 gene (e.g., a mutant PNPLA3 gene that causes amyloid deposition), or a transgenic PNPLA3 gene in relation to genetically modified cells, cell populations, or organisms.

[0419] "Inhibiting PNPLA3 gene expression" includes inhibition of any level of PNPLA3 gene expression, e.g., suppression of at least a portion of PNPLA3 gene expression. PNPLA3 gene expression can be assessed based on the level or change in level of any variable associated with PNPLA3 gene expression, such as PNPLA3 mRNA levels, PNPLA3 protein levels, or the number or extent of amyloid deposits. This level can be assessed in individual cells or cell populations, including, for example, samples derived from the subject.

[0420] Inhibition may be assessed by a reduction in the absolute or relative level of one or more variables related to PNPLA3 expression compared to a control level. The control level may be any type of control level used in the art, such as the baseline level before administration, or a level determined from an untreated or treated similar subject, cell, or sample (e.g., a buffer-only control or an inactivator control).

[0421] In some embodiments of the method of the present invention, PNPLA3 gene expression is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99%.

[0422] Inhibition of PNPLA3 gene expression can be manifested by a decrease in the amount of mRNA expressed by the first cells or cell population (such cells may be present, for example, in a sample derived from the subject) that have been transcribed and treated (e.g., by contacting the cells with the RNAi agent of the present invention, or by administering the RNAi agent of the present invention to a subject in which such cells are present or have been present) to inhibit PNPLA3 gene expression, which is substantially identical to that of the first cells or cell population, but is similarly identical to that of the second cells or cell population (control cells) that have not been treated in the same manner. In a preferred embodiment, the inhibition is expressed by the following formula:

number

[0423] Alternatively, inhibition of PNPLA3 gene expression can be evaluated in terms of a decrease in parameters functionally related to PNPLA3 gene expression, such as PNPLA3 protein expression or hedgehog pathway protein activity. PNPLA3 gene silencing can be determined constitutively or by genomically engineered cell culture by any assay known in the art.

[0424] Inhibition of PNPLA3 protein expression can be manifested by a decrease in the level of PNPLA3 protein expressed by cells or cell populations (e.g., the level of protein expressed in a sample derived from the target). As described above, for the evaluation of mRNA suppression, inhibition of protein expression levels in treated cells or cell populations can be expressed similarly as a percentage of the protein level in control cells or cell populations.

[0425] Control cells or cell populations that can be used to evaluate the inhibition of PNPLA3 gene expression include cells or cell populations that have not yet been exposed to the RNAi agent of the present invention. For example, control cells or cell populations can be obtained from individual subjects (e.g., human or animal subjects) before treatment of the subject with the RNAi agent.

[0426] The level of PNPLA3 mRNA expressed by a cell or cell population, or the level of circulating PNPLA3 mRNA, can be determined using any method known in the art for evaluating mRNA expression. In one embodiment, the expression level of PNPLA3 in a sample is determined, for example, by detecting a transcription polynucleotide, such as the mRNA of the PNPLA3 gene, or a portion thereof. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats using ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating PNPLA3 mRNA can be detected using the method described in PCT / US2012 / 043584, the full contents of which are incorporated herein by reference.

[0427] In one embodiment, the expression level of PNPLA3 is determined using a nucleic acid probe. The term "probe," as used herein, refers to any molecule capable of selectively binding to a particular PNPLA3. Probes can be synthesized by those skilled in the art or obtained from suitable biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0428] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern blot analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with PNPLA3 mRNA. In one embodiment, for example, the isolated mRNA is electrophoresed on an agarose gel, and the mRNA is immobilized on a solid surface by transferring it from the gel to a membrane such as nitrocellulose, and then contacted with the probe. In another embodiment, the probe is immobilized on a solid surface using, for example, an Affymetrix gene chip array, and the mRNA is contacted with the probe. Those skilled in the art can easily adapt known mRNA detection methods for use in determining PNPLA3 mRNA levels.

[0429] Other methods for determining the expression level of PNPLA3 in a sample include, for example, RT-PCR (experimental embodiments described in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), autologous persistent sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology 6:1197), and rolling circle replication (Lizardi et al. The method includes, for example, the amplification of mRNA in a sample and / or the detection of the amplified molecule using techniques well known to those skilled in the art, after the steps of nucleic acid amplification and / or reverse transcriptase (for generating cDNA) by any other nucleic acid amplification method (e.g., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method. These detection schemes are particularly useful for detecting nucleic acid molecules when very few nucleic acid molecules are present. In specific embodiments of the present invention, the expression level of PNPLA3 is quantified by quantitative fluorescence RT-PCR (i.e., the TaqMan® System).

[0430] The expression level of PNPLA3 mRNA can be monitored using a membrane blot (e.g., those used in hybridization analyses such as Northern, Southern, and Dot), or using microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patents No. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determination of PNPLA3 expression levels may also include the use of nucleic acid probes in solution.

[0431] In preferred embodiments, mRNA levels are assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of these methods is described and illustrated in the examples provided herein.

[0432] The level of PNPLA3 protein expression can also be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high-diffusion chromatography, liquid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (one- or two-way), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosolvent assay (ELISA), immunofluorescence assay, and electrochemiluminescence assay.

[0433] In some embodiments, the effects of the method of the present invention can be monitored by detecting or monitoring the reduction of symptoms of PNPLA3 disease, such as edematous swelling of the limbs, face, larynx, upper respiratory tract, abdomen, trunk, and genitals; prodromal symptoms; laryngeal edema; non-pruritic rash; nausea; vomiting; or abdominal pain. These symptoms can be evaluated in vitro or in vivo using any method known in the art.

[0434] In some embodiments of the method of the present invention, an RNAi agent is administered to a subject so that the RNAi agent is delivered to a specific site within the subject. Inhibition of PNPLA3 expression can be evaluated by measuring the level or change therein of PNPLA3 mRNA or PNPLA3 protein in a sample obtained from body fluid or tissue from a specific site within the subject. In preferred embodiments, the above site is selected from the group consisting of the liver, choroid plexus, retina, and pancreas. The above site may be a small unit or subgroup of cells from any one of the above-mentioned sites. This site may also include cells expressing a specific receptor.

[0435] VIII. Methods for treating or preventing PNPLA3-related disorders The present invention provides a method for the treatment and prevention of PNPLA3-related diseases, disorders, and / or conditions, comprising the step of administering a composition containing an iRNA agent, a pharmaceutical composition containing an iRNA agent, or a vector containing the iRNA of the present invention to a subject having or being prone to developing PNPLA3-related diseases, disorders, and / or conditions. Non-limiting examples of PNPLA3-related diseases include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, intrahepatic fat accumulation, hepatic inflammation, hepatocyte necrosis, hepatic fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). In one embodiment, the PNPLA3-related disease is NAFLD. In another embodiment, the PNPLA3-related disease is NASH. In another embodiment, the PNPLA3-related disease is fatty liver (steatosis). In another embodiment, the PNPLA3-related disease is insulin resistance. In another embodiment, the PNPLA3-related disease is not insulin resistance.

[0436] The methods of the present invention are useful in treating subjects with PNPLA3-related diseases, such as subjects who would benefit from reduced PNPLA3 gene expression and / or PNPLA3 protein production. In one embodimen...

Claims

1. A double-stranded ribonucleic acid (RNAi) agent or a salt thereof for inhibiting the expression of patatin-like phospholipase domain 3 (PNPLA3), The double-stranded RNAi agent or a salt thereof comprises a sense strand and an antisense strand that form a double-stranded region of 30 nucleotides or less in length. The sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence 5'-UCUGAGCUGAGUGUUGGUUUUAUAU-3' of SEQ ID NO: 1258, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence 5'-AUAAAACCAACUCACAGCUCAGAGG-3' of SEQ ID NO: 1348. The antisense strand is 15-23 nucleotides long. All of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include nucleotide modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, and The aforementioned at least one chain is conjugated to a ligand. Double-stranded RNAi agents or their salts.

2. The double-stranded RNAi agent or a salt thereof according to claim 1, wherein the antisense strand comprises at least 17 consecutive nucleotides from the nucleotide sequence 5'-AUAAAACCAACUCACAGUCAGAGAG-3' of SEQ ID NO: 1348.

3. The double-stranded RNAi agent or a salt thereof according to claim 2, wherein the antisense strand comprises at least 19 consecutive nucleotides from the nucleotide sequence 5'-AUAAAACCAACUCACAGUCAGAGAG-3' of SEQ ID NO: 1348.

4. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein the sense strand comprises at least 17 consecutive nucleotides from the nucleotide sequence 5'-UCUGAGCUGAGUUGGUUUUAUAU-3' of SEQ ID NO: 1258.

5. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein the sense strand comprises at least 19 consecutive nucleotides from the nucleotide sequence 5'-UCUGAGCUGAGUUGGUUUUAUAU-3' of SEQ ID NO: 1258.

6. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein the sense strand is 15 to 30 nucleotides long.

7. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein the antisense strand is 19 to 23 nucleotides long.

8. The double-stranded RNAi agent or a salt thereof according to claim 7, wherein the antisense strand is 21 to 23 nucleotides long.

9. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

10. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

11. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein the ligand comprises GalNAc.

12. The double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein the ligand comprises one or more GalNAc derivatives bound via a divalent or trivalent branched linker.

13. The ligand is 【Chemistry 1】 The double-stranded RNAi agent or a salt thereof according to claim 12.

14. The double-stranded RNAi agent or a salt thereof according to claim 11, wherein the ligand is bound to the 3' end of the sense strand.

15. The double-stranded RNAi agent or its salt is shown in the following schematic diagram: 【Chemistry 2】 A double-stranded RNAi agent or a salt thereof according to claim 14, which is conjugated to a ligand shown in [reference].

16. The double-stranded RNAi agent or salt thereof according to any one of claims 1 to 3, wherein the double-stranded RNAi agent or salt thereof further comprises at least one phosphorothioate or methylphosphonate internucleotide bond.

17. The double-stranded RNAi agent or a salt thereof according to claim 16, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 3' end of one of the strands.

18. The double-stranded RNAi agent or a salt thereof according to claim 17, wherein the aforementioned strand is the antisense strand.

19. The double-stranded RNAi agent or a salt thereof according to claim 17, wherein the aforementioned strand is the sense strand.

20. The double-stranded RNAi agent or a salt thereof according to claim 16, wherein the phosphorothioate or methylphosphonate internucleotide bond is located at the 5' end of one of the strands.

21. The double-stranded RNAi agent or a salt thereof according to claim 20, wherein the aforementioned strand is the antisense strand.

22. The double-stranded RNAi agent or a salt thereof according to claim 20, wherein the aforementioned strand is the sense strand.

23. The double-stranded RNAi agent or salt thereof according to claim 21, wherein the phosphorothioate or methylphosphonate internucleotide bond is present at both the 5' and 3' ends of one of the strands.

24. The double-stranded RNAi agent or a salt thereof according to claim 23, wherein the aforementioned strand is the antisense strand.

25. The double-stranded RNAi agent or salt thereof according to any one of claims 1 to 3, wherein the double-stranded RNAi agent or salt thereof further comprises a 6-8 phosphorothioate or methylphosphonate internucleotide bond.

26. A double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 3, wherein the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

27. A double-stranded ribonucleic acid (RNAi) agent or a salt thereof for inhibiting PNPLA3 expression, The double-stranded RNAi agent or a salt thereof comprises a sense strand and an antisense strand that form a double-stranded region of 30 nucleotides or less in length. The sense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence 5'-UCUGAGCUGAGUGUUGGUUUUAUAU-3' of SEQ ID NO: 1258, and the antisense strand comprises at least 15 consecutive nucleotides from the nucleotide sequence 5'-AUAAAACCAACUCACAGCUCAGAGG-3' of SEQ ID NO: 1348. The antisense strand is 15-23 nucleotides long. All of the nucleotides of the sense strand and 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 double-stranded RNAi agent or a salt thereof comprises a 6-8 phosphorothioate or methylphosphonate internucleotide bond, and The aforementioned sense strand is conjugated to a ligand containing GalNAc. Double-stranded RNAi agents or their salts.

28. The double-stranded RNAi agent or a salt thereof according to claim 27, wherein the antisense strand is 19 to 23 nucleotides long.

29. The double-stranded RNAi agent or a salt thereof according to claim 27 or 28, wherein the sense strand is 15 to 30 nucleotides long.

30. Cells containing a double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 29.

31. A pharmaceutical composition for inhibiting the expression of the PNPLA3 gene, comprising a double-stranded RNAi agent or a salt thereof as described in any one of claims 1 to 29.

32. The pharmaceutical composition according to claim 31, wherein the double-stranded RNAi agent or a salt thereof is present in a non-buffer.

33. The pharmaceutical composition according to claim 32, wherein the non-buffering agent is saline solution or water.

34. The pharmaceutical composition according to claim 31, wherein the double-stranded RNAi agent or a salt thereof is present in the buffer.

35. The pharmaceutical composition according to claim 34, wherein the buffer solution comprises an acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

36. The pharmaceutical composition according to claim 35, wherein the buffer solution is phosphate-buffered saline (PBS).

37. An in vitro method for inhibiting PNPLA3 expression in cells, (a) the step of bringing the cells into contact with a double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 29 or a pharmaceutical composition according to any one of claims 31 to 36; and (b) Maintaining the cells generated in step (a) for a sufficient amount of time to achieve degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting the expression of the PNPLA3 gene in the cells. In vitro methods including those mentioned above.

38. The method according to claim 37, wherein the expression of PNPLA3 is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%.

39. A pharmaceutical composition according to any one of claims 31 to 36, comprising a therapeutically effective amount of a double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 29, for treating a subject having a disorder that benefits from reduced PNPLA3 expression.

40. A pharmaceutical composition according to any one of claims 31 to 36, comprising a prophylactic effective amount of a double-stranded RNAi agent or a salt thereof according to any one of claims 1 to 29, for preventing at least one symptom of a subject having a disorder that benefits from reduced PNPLA3 expression.

41. The pharmaceutical composition according to claim 39 or 40, wherein the disorder is a PNPLA3-related disorder.

42. The pharmaceutical composition according to claim 41, wherein the PNPLA3-related disease is non-alcoholic fatty liver disease (NAFLD).

43. The pharmaceutical composition according to claim 41, wherein the PNPLA3-related disease is fatty liver (steatosis).

44. The pharmaceutical composition according to claim 41, wherein the PNPLA3-related disease is non-alcoholic steatohepatitis (NASH).

45. The pharmaceutical composition according to any one of claims 39 to 44, wherein the subject is a human.

46. A pharmaceutical composition according to any one of claims 39 to 45, administered together with an anti-PNPLA3 antibody or its antigen-binding fragment.

47. The pharmaceutical composition according to any one of claims 39 to 46, wherein the double-stranded RNAi agent or a salt thereof is for subcutaneous administration.

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