Tmprss6 irna compositions and methods of use thereof

Abstract

To provide a method for effective treatment of an iron overload associated disorder.SOLUTION: The present invention provides RNAi agents, e.g., double-stranded RNAi agents targeting the TMPRSS6 gene, and methods of using such RNAi agents to inhibit expression of TMPRSS6 and methods of treating subjects having a TMPRSS6 associated disorder, e.g., an iron overload associated disorder, such as β-thalassemia or hemochromatosis.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 61 / 826,178, filed May 22, 2013, and U.S. Provisional Patent Application No. 61 / 912,988, filed December 6, 2013. This application is related to U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, and PCT / US2012 / 065601, filed November 16, 2012. The entire contents of each of the above provisional patent applications are hereby incorporated by reference into this specification.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The name of the ASCII copy created on May 21, 2014 is 121301-00720_SL.txt, and the size is 449,620 bytes.

Background Art

[0003] The TMPRSS6 (transmembrane protease, serine 6) gene encodes TMPRSS6, also known as matriptase-2, a type II serine protease. The TMPRSS6 gene is mainly expressed in the liver, but high levels of TMPRSS6 mRNA are also seen in the kidney, lower levels are seen in the uterus, and much smaller amounts are detected in many other tissues (Non-Patent Document 1). TMPRSS6 binds to the hepcidin activator and BMP coreceptor HJV (hemojuvelin), degrades the protein, and thereby plays a role in iron homeostasis by causing downregulation of hepcidin levels.

[0004] TMPRSS6 consists of a short N-terminal cytoplasmic tail, a type II transmembrane domain, a stem region composed of two extracellular CUB (complement factor Cls / Clr, sea urchin embryonic growth factor, and BMP (bone morphogenetic protein)) domains, three LDLR (low density lipoprotein receptor class A) domains, and a C-terminal trypsin-like serine protease domain. There are also consensus sites for N-glycosylation in the extracellular domain and potential phosphorylation sites in the cytoplasmic tail region.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Many diseases may be associated with iron overload, a pathological condition characterized by increased levels of iron. Iron overload can cause excessive iron deposition in various tissues, which can lead to tissue and organ damage. Therefore, there is a current need for methods for the effective treatment of diseases associated with iron overload.

Means for Solving the Problems

[0007] The present invention provides a composition comprising an RNAi agent targeting TMPRSS6, for example, a double-stranded iRNA agent. The present invention also provides a method of using the composition of the present invention for treating diseases associated with TMPRSS6 expression inhibition, such as diseases associated with iron overload, such as thalassemia, such as β-thalassemia, or hemochromatosis.

[0008] Accordingly, in one aspect, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (matriptase-2) in cells, for example, a double-stranded RNAi agent, 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 are identical to any one of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 with three or fewer nucleotides different therefrom, and the antisense strand comprising at least 15 consecutive nucleotides that are identical to any one of the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 with three or fewer nucleotides different therefrom. Substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. There is provided a double-stranded RNAi agent in which the sense strand is conjugated to a ligand bound at the 3'-end.

[0009] In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0010] In one embodiment, the sense strand and the antisense strand comprise a complementary region comprising at least 15 consecutive nucleotides that are identical to any one of the antisense sequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12 with three or fewer nucleotides different therefrom.

[0011] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-basic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-phosphate or a 5'-phosphate mimetic (see, e.g., PCT Publication No. WO 2011 / 005860), and a terminal nucleotide linked to a cholesteryl derivative or a didodecylamide group of dodecanoic acid).

[0012] In one embodiment, at least one strand comprises a 3'-overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3'-overhang of at least two nucleotides. In another aspect, the present invention is an RNAi agent capable of inhibiting the expression of TMPRSS6 (matriptase-2) in cells, e.g., a double-stranded RNAi agent, 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 TMPRSS6, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’(III) (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 independently containing either modified or unmodified 0 to 25 nucleotides or combinations thereof, with each sequence containing at least two different modified nucleotides; each N b and N b ’ represents an oligonucleotide sequence independently containing either modified or unmodified 0 to 10 nucleotides or combinations thereof; each n, which may or may not be present, p , n p ’, n q , and n q ’ independently represents an overhang nucleotide; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent one motif of three identical modifications on three consecutive nucleotides; The modification on N b is different from the modification on Y, and the modification on N b ’ is different from the modification on Y’; The sense strand is conjugated to at least one ligand) to provide a double-stranded RNAi agent represented by.

[0013] 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.

[0014] In one embodiment, XXX is complementary to X’X’X’, YYY is complementary to Y’Y’Y’, and ZZZ is complementary to Z’Z’Z’.

[0015] In one embodiment, the YYY motif is present at or near the cleavage site of the sense strand.

[0016] In one embodiment, the Y’Y’Y’ motif is present at positions 11, 12, and 13 of the antisense strand at the 5’ end.

[0017] In one embodiment, Y’ is 2’-O-methyl.

[0018] In one embodiment, formula (III) is formula (IIIa): Sense: 5’n p -N a -YYY-N a -n q 3’ Antisense: 3’n p’ -N a’ -Y’Y’Y’-N a’ -n q’ 5’(IIIa) represented by.

[0019] In another embodiment, formula (III) is formula (IIIb): Sense: 5’n p -N a -YYY-N b -ZZZ-N a -n q 3’ Antisense: 3’n p’ -N a’ -Y’Y’Y’-N b’ -Z’Z’Z’-N a’ -n q’ 5’(IIIb) (where each N b and N b ’ independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) represented by.

[0020] In yet another embodiment, formula (III) is formula (IIIc): Sense: 5’n p -N a -XXX-N b -YYY-Na -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5'(IIIc) (wherein each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) is represented by.

[0021] In one embodiment, formula (III) is formula (IIId): Sense: 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense: 3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIId) (wherein each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N a and N a ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides) is represented by.

[0022] In one embodiment, the double-stranded region is 15 to 30 nucleotide pairs in length. In another embodiment, the double-stranded region is 17 to 23 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 17 to 25 nucleotide pairs in length. In one embodiment, the double-stranded region is 23 to 27 nucleotide pairs in length. In another embodiment, the double-stranded region is 19 to 21 nucleotide pairs in length. In another embodiment, the double-stranded region is 21 to 23 nucleotide pairs in length. In one embodiment, each strand has 15 to 30 nucleotides. In another embodiment, each strand has 19 to 30 nucleotides.

[0023] In one embodiment, the modification on the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In another embodiment, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0024] In one embodiment, the ligand is one or more GalNAc derivatives conjugated via a divalent or trivalent branched linker. In another embodiment, the ligand is

Chemical formula

[0025] In one embodiment, the ligand is attached to the 3'-end of the sense strand.

[0026] In one embodiment, the RNAi agent is conjugated to a ligand represented by the following schematic diagram

Chemical formula

[0027] In one embodiment, the agent further comprises at least one phosphorothioate or methylphosphonate nucleotide internucleotide linkage.

[0028] In one embodiment, the phosphorothioate or methylphosphonate nucleotide internucleotide linkage is at the 3' end of one strand. In one embodiment, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

[0029] In one embodiment, the phosphorothioate or methylphosphonate nucleotide internucleotide linkage is at the 5' end of one strand. In one embodiment, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

[0030] In one embodiment, the phosphorothioate or methylphosphonate nucleotide internucleotide linkage is at both the 5' and 3' ends of one strand. In one embodiment, the strand is an antisense strand.

[0031] In one embodiment, the RNAi agent comprises 6 to 8 phosphorothioate nucleotide internucleotide linkages.

[0032] In one embodiment, the antisense strand comprises two phosphorothioate nucleotide internucleotide linkages at the 5' end and two phosphorothioate nucleotide internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate nucleotide internucleotide linkages at either the 5' or 3' end.

[0033] In one embodiment, the base pair at position 1 at the 5' end of the double-stranded antisense strand is an AU base pair.

[0034] In one embodiment, the Y nucleotide comprises a 2'-fluoro modification.

[0035] In one embodiment, the Y' nucleotide comprises a 2'-O-methyl modification.

[0036] In one embodiment, p’ > 0. In another embodiment, p’ = 2.

[0037] In one 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 non - complementary 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.

[0040] In one embodiment, all n p ’ are linked to an adjacent nucleotide via a phosphorothioate bond.

[0041] In one embodiment, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12.

[0042] In one embodiment, the RNAi agent is AD - 59743. In another embodiment, the RNAi agent is AD - 60940.

[0043] In one aspect, the present invention is a double - stranded RNAi agent for inhibiting the expression of TMPRSS6 in a cell, wherein the double - stranded RNAi agent comprises a sense strand and an antisense strand that form a double - stranded region, The sense strand contains at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 by three or fewer nucleotides, and the antisense strand contains at least 15 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 by three or fewer nucleotides. Substantially all of the nucleotides of the sense strand contain a modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification. The sense strand contains two phosphorothioate internucleotide linkages at the 5' end. Substantially all of the nucleotides of the antisense strand contain a modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification. The antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end. The present invention provides a double-stranded RNAi agent in which the sense strand is conjugated to one or more GalNAc derivatives linked via a branched bivalent or trivalent linker at the 3' end.

[0044] In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain a modification.

[0045] In another aspect, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (matriptase-2) in a cell, for example, a double-stranded RNAi agent, wherein the double-stranded RNAi agent contains a sense strand complementary to the antisense strand, the antisense strand contains a region complementary to a part of the mRNA encoding TMPRSS6, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -Na -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’(III) (wherein: i, j, k, and l are each independently 0 or 1; p, p’, q, and q’ are each independently 0 to 6; each N a and N a ’ represents an oligonucleotide sequence containing either modified or unmodified 0 to 25 nucleotides or combinations thereof, with each sequence containing at least two different modified nucleotides; each N b and N b ’ represents an oligonucleotide sequence containing either modified or unmodified 0 to 10 nucleotides or combinations thereof; each n p , n p ’, n q , and n q ’ independently represent overhang nucleotides; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent one motif of three identical modifications on three consecutive nucleotides, where the modification is a 2’-O-methyl or 2’-fluoro modification; the modification on N b is different from the modification on Y, and the modification on N b ’ is different from the modification on Y’; (the sense strand is conjugated to at least one ligand) to provide a double-stranded RNAi agent represented by.

[0046] In yet another aspect, the present invention is an RNAi agent capable of inhibiting the expression of TMPRSS6 (matriptase-2) in cells, for example, a double-stranded RNAi agent, 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 TMPRSS6, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’(III) (Wherein: i, j, k, and l are each independently 0 or 1; Each n that may or may not be present p , n q , and n q ’ independently represents an overhang nucleotide; p, q, and q’ are each independently 0 to 6; n p ’>0, and at least one n p ’ is linked to an adjacent nucleotide via a phosphorothioate bond; Each N a and N a ’ independently represents an oligonucleotide sequence comprising either modified or unmodified 0 to 25 nucleotides or a combination thereof, and each sequence comprises at least two different modified nucleotides; Each N b and N b‘ independently represents an oligonucleotide sequence comprising either a modified or unmodified 0 to 10 nucleotide or a combination thereof; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent one motif of three identical modifications on three consecutive nucleotides, and the modification is a 2’-O-methyl or 2’-fluoro modification; N b The modification above is different from the modification on Y, N b The modification on ‘ is different from the modification on Y’; (the sense strand is conjugated to at least one ligand) Provided is a double-stranded RNAi agent represented by.

[0047] In a further aspect, the present invention relates to an RNAi agent capable of inhibiting the expression of TMPRSS6 (matriptase-2) in cells, for example, a double-stranded RNAi agent, 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 TMPRSS6, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’(III) (wherein: i, j, k, and l are each independently 0 or 1; each n which may or may not be present p , nq and n q ’ independently represents an overhanging nucleotide; p, q, and q’ are each independently 0 to 6; n p ’ > 0, and at least one n p ’ is linked to an adjacent nucleotide via a phosphorothioate bond; each N a and N a ’ independently represents an oligonucleotide sequence comprising from 0 to 25 nucleotides, either modified or unmodified, or a combination thereof, each sequence comprising at least two different modified nucleotides; each N b and N b ’ independently represents an oligonucleotide sequence comprising from 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent one motif of three identical modifications on three consecutive nucleotides, the modification being a 2’-O-methyl or 2’-fluoro modification; N b the modification on N is different from the modification on Y, and the modification on N b ’ is different from the modification on Y’; The sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives conjugated via a divalent or trivalent branched linker) to provide a double-stranded RNAi agent represented by.

[0048] In another aspect, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (matriptase-2) in cells, for example, a double-stranded RNAi agent, 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 TMPRSS6, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -Na -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’-N a ’-(X’X’X’) k -N b ’-Y’Y’Y’-N b ’-(Z’Z’Z’) l -N a ’-n q ’5’(III) (wherein: i, j, k, and l are each independently 0 or 1; each n, which may or may not be present, p , n q , and n q ’ independently represents an overhang nucleotide; p, q, and q’ are each independently 0 to 6; n p ’ > 0, and at least one n p ’ is linked to an adjacent nucleotide via a phosphorothioate bond; each N a and N a ’ independently represents an oligonucleotide sequence comprising either modified or unmodified 0 to 25 nucleotides or combinations thereof, each sequence containing at least two different modified nucleotides; each N b and N b ’ independently represents an oligonucleotide sequence comprising either modified or unmodified 0 to 10 nucleotides or combinations thereof; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent one motif of three identical modifications on three consecutive nucleotides, the modification being a 2’-O-methyl or 2’-fluoro modification; N b The modification on N is different from the modification on Y, N bThe modification on the sense strand is different from the modification on the antisense strand; The sense strand contains at least one phosphorothioate bond; The sense strand is conjugated to at least one ligand, and the ligand is one or more GalNAc derivatives conjugated via a divalent or trivalent branched linker) There is provided a double-stranded RNAi agent represented by the following formula.

[0049] In yet another aspect, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (matriptase-2) in cells, for example, a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, the antisense strand contains a region complementary to a part of the mRNA encoding TMPRSS6, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5’n p -N a -YYY-N a -n q 3’ Antisense: 3’n p ’-N a ’-Y’Y’Y’-N a ’-n q ’5’ (IIIa) (In the formula: Each n, which may or may not be present, p n q and n q ’ represents an overhang nucleotide independently; p, q, and q’ are each independently 0 to 6; n p ’>0, and at least one n p ’ is bound to an adjacent nucleotide via a phosphorothioate bond; Each N a and N a ’ represents an oligonucleotide sequence containing either modified or unmodified 0 to 25 nucleotides or a combination thereof independently, and each sequence contains at least two different modified nucleotides; YYY and Y’Y’Y’ each independently represent one motif of three identical modifications on a triple nucleotide, and the modification is a 2’-O-methyl or 2’-fluoro modification; The sense strand contains at least one phosphorothioate bond; The sense strand is conjugated to at least one ligand, and the ligand is one or more GalNAc derivatives conjugated via a divalent or trivalent branched linker) Provided is a double-stranded RNAi agent represented by.

[0050] In one embodiment, the present invention provides an RNAi agent selected from the group of RNAi agents listed in any one of Tables 1, 2, 4, 5, 8, 19, and 12.

[0051] In one aspect, the present invention is a composition comprising a modified antisense polynucleotide agent, the agent being capable of inhibiting the expression of TMPRSS6 intracellularly, and comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12, and the polynucleotide having a length of about 14 to about 30 nucleotides.

[0052] The present invention also provides cells, vectors, host cells, and pharmaceutical compositions comprising, for example, the double-stranded RNAi agent of the present invention.

[0053] In certain embodiments, the RNAi agent is administered using a pharmaceutical composition.

[0054] In a preferred embodiment, the RNAi agent is administered in solution. In certain such embodiments, the siRNA is administered in a non-buffered solution. In one embodiment, the siRNA is administered in water. In other embodiments, the siRNA is administered with a buffer such as an acetate buffer, a citrate buffer, a prolamine buffer, a carbonate buffer, or a phosphate buffer or any combination thereof. In certain embodiments, the buffer is phosphate buffered saline (PBS).

[0055] In one embodiment, the pharmaceutical composition further comprises a lipid formulation. In one embodiment, the lipid formulation comprises an LNP or an XTC. In another embodiment, the lipid formulation comprises an MC3.

[0056] In one aspect, the present invention provides a method for inhibiting TMPRSS6 expression in a cell. The method comprises contacting the cell with an RNAi agent, such as a double-stranded RNAi agent, or a modified antisense polynucleotide agent of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the TMPRSS6 gene, thereby inhibiting the expression of the TMPRSS6 gene in the cell.

[0057] In one embodiment, the cell is in a subject.

[0058] In one embodiment, the subject is human.

[0059] In one embodiment, TMPRSS6 expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%.

[0060] In another embodiment, hepcidin gene expression is increased by at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, or about 5-fold.

[0061] In yet another embodiment, the serum hepcidin concentration is increased by at least about 10%, about 25%, about 50%, about 100%, about 150%, about 200%, about 250%, or about 300%.

[0062] In one embodiment, the serum iron concentration is decreased by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0063] In another embodiment, the transferrin saturation percentage is decreased by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0064] In another aspect, the present invention provides a method of treating a subject suffering from a disease mediated by or associated with TMPRSS6 expression. The method includes administering to the subject a therapeutically effective amount of an RNAi agent of the present invention, such as a double-stranded RNAi agent, or a modified antisense polynucleotide agent of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby treating the subject.

[0065] In one aspect, the present invention provides a method of treating a subject suffering from a disease associated with TMPRSS6. The method includes subcutaneously administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, thereby treating the subject, wherein the double-stranded RNAi agent includes a sense strand and an antisense strand that form a double-stranded region, the sense strand includes at least 15 consecutive nucleotides that are identical to any one of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 with no more than 3 nucleotides different, and the antisense strand includes at least 15 consecutive nucleotides that are identical to any one of the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 with no more than 3 nucleotides different, substantially all of the nucleotides of the antisense strand include a modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification, the antisense strand includes two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, substantially all of the nucleotides of the sense strand include a modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification, the sense strand includes two phosphorothioate internucleotide linkages at the 5' end, The sense strand is conjugated to one or more GalNAc derivatives linked via a branched divalent or trivalent linker at the 3'-end.

[0066] In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include modifications.

[0067] In one embodiment, the subject is human.

[0068] In one embodiment, the subject suffers from a disease associated with iron overload, such as hereditary hemochromatosis, β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia), erythropoietic porphyria, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia.

[0069] In one embodiment, the RNAi agent, such as a double-stranded RNAi agent, is administered at a dose of about 0.01 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 10 mg / kg, about 4 mg / kg to about 10 mg / kg, about 5 mg / kg to about 15 mg / kg, about 6 mg / kg to about 15 mg / kg, about 7 mg / kg to about 15 mg / kg, about 8 mg / kg to about 15 mg / kg, about 9 mg / kg to about 15 mg / kg, about 10 mg / kg to about 20 mg / kg, about 12 mg / kg to about 20 mg / kg, about 13 mg / kg to about 20 mg / kg, about 14 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 16 mg / kg to about 20 mg / kg, or about 18 mg / kg to about 20 mg / kg. In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 0.1 mg / kg, about 1.0 mg / kg, or about 3.0 mg / kg.

[0070] In one embodiment, the RNAi agent, such as a double-stranded RNAi agent, is administered subcutaneously or intravenously.

[0071] In one embodiment, the RNAi agent is administered in two or more doses. In certain embodiments, the RNAi agent is administered at intervals selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, about once every 96 hours, about once every 7 days, or about once every 14 days. In certain embodiments, the RNAi agent is administered once a week for up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5 weeks, or longer.

[0072] In yet another aspect, the present invention provides a method of treating a disease associated with iron overload in a subject. The method includes administering to the subject a therapeutically effective amount of an RNAi agent, such as a double-stranded RNAi agent, or a vector of the present invention, thereby treating the subject.

[0073] In one embodiment, the disease associated with iron overload is hemochromatosis. In another embodiment, the disease associated with iron overload is thalassemia, such as β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia), or erythropoietic porphyria. In yet another embodiment, the disease associated with iron overload is a neurological disease, such as Parkinson's disease, Alzheimer's disease or Friedreich's ataxia.

[0074] In one embodiment, the subject is a primate or a rodent. In another embodiment, the subject is a human.

[0075] In one embodiment, the RNAi agent, such as a double-stranded RNAi agent, is administered at a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0076] In one embodiment, the RNAi agent, such as a double-stranded RNAi agent, is administered subcutaneously or intravenously.

[0077] In one embodiment, the RNAi agent is administered at two or more doses. In certain embodiments, the RNAi agent is administered at intervals selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, about once every 96 hours, about once every 7 days, or about once every 14 days.

[0078] In one embodiment, administration results in a decrease in iron levels, ferritin levels, and / or transferrin saturation levels in the subject.

[0079] In one embodiment, the method further comprises determining the iron level in the subject.

[0080] In one embodiment, the method of the invention comprising administering the iRNA agent (or the pharmaceutical composition of the invention) to a subject is practiced in combination with administration of additional agents and / or other methods of treatment. In one embodiment, the method of the invention further comprises administering to the subject an iron chelating agent, such as deferiprone, deferoxamine, and deferasirox.

[0081] The present invention is further illustrated by the following detailed description and drawings.

Brief Description of the Drawings

[0082]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0083] The present invention provides an RNAi agent targeting TMPRSS6, for example, a composition containing a double-stranded iRNA agent. The present invention also provides a method of using the composition of the present invention for inhibiting TMPRSS6 expression and treating diseases associated with TMPRSS6, such as β-thalassemia or hemochromatosis.

[0084] TMPRSS6 plays an important role in iron homeostasis as an inhibitor of HAMP gene expression. The HAMP gene encodes the liver hormone hepcidin, a central regulator of iron homeostasis. Hepcidin binds to the iron exporter protein ferroportin (FPN1), which is mainly localized in absorptive enterocytes, hepatocytes, and macrophages. Hepcidin binding to the extracellular domain of ferroportin leads to the internalization and degradation of ferroportin, thus reducing the absorption of dietary iron from the intestine and the release of iron from macrophages and hepatocytes. HAMP gene expression can be stimulated in response to iron through a bone morphogenetic protein (BMP) / Sons of Mothers Against Decapentaplegic (SMAD)-dependent signaling cascade mediated by the BMP coreceptor hemojuvelin (HJV). The important role of TMPRSS6 in HAMP regulation is the inhibition of BMP-mediated HAMP upregulation. TMPRSS6 cleaves the BMP coreceptor HJV, which is essential for BMP-mediated HAMP upregulation; thus, it inhibits BMP-mediated HAMP upregulation by preventing BMP signaling, SMAD translocation to the nucleus, and HAMP transcriptional activation.

[0085] Several human and mouse studies have confirmed the role of TMPRSS6 in HAMP regulation and iron homeostasis (Du et al. Science 2008, Vol. 320, pp1088 - 1092; Folgueras et al. Blood 2008, Vol. 112, pp2539 - 45). Studies have shown that loss-of-function mutations in TMPRSS6 result in upregulation of hepcidin expression, characterized by elevated hepcidin levels, hypochromic microcytic anemia, low mean corpuscular volume (MCV), low transferrin saturation, inadequate oral iron absorption, and an incomplete response to parenteral iron, and can cause a hereditary iron deficiency anemia called iron-refractory iron deficiency anemia (IRIDA) (Finberg. Seminars in Hematology 2009, Vol. 46, pp378 - 86). However, loss-of-function mutations in positive regulators of HAMP (e.g., BMP1, BMP4, and HFE) have been shown to downregulate hepcidin expression and cause iron overload (Milet et al. Am J Hum Gen 2007, Vol. 81, pp799 - 807; Finberg et al. Blood 2011, Vol. 117, pp4590 - 9). In primary iron overload, collectively called hereditary hemochromatosis (HH), anemia characterized by a large amount of ineffective hematopoiesis, and iron overload (secondary hemochromatosis) such as intermediate β-thalassemia (TI), hepcidin levels are low despite elevated serum iron concentrations and iron stores. A mouse model of intermediate β-thalassemia demonstrated that decreased TMPRSS6 expression results in increased hepcidin levels (Finberg 2010 Oral Presentation: “TMPRSS6, an inhibitor of Hepatic BMP / Smad Signaling, is required for Hepcidin Suppression and Iron Loading in a Mouse Model of β-Thalassemia”. American Society of Hematology Annual Meeting 2010, Abstract No.: 164).

[0086] The present invention describes iRNA agents, compositions and methods for regulating the expression of the TMPRSS6 gene. In certain embodiments, the expression of TMPRSS6 is reduced or inhibited using a TMPRSS6-specific iRNA agent, thereby resulting in an increase in HAMP expression and a decrease in serum iron levels. Thus, inhibition of the expression or activity of the TMPRSS6 gene using the iRNA compositions contemplated in the present invention can be a useful approach for the treatment aimed at reducing iron levels in a subject. Such inhibition can be useful for treating diseases associated with iron overload such as hemochromatosis or thalassemia, for example, β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia).

[0087] I. Definitions To make the present invention more readily understandable, some terms are first defined. Further, it should be noted that whenever a value or range of values of a variable is recited, values intermediate to and within the recited ranges are also intended to be part of the present invention.

[0088] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0089] The term "including" is used herein to mean the phrase "including but not limited to" and is used synonymously with this phrase.

[0090] The term "or" is used herein to mean the term "and / or" unless the context clearly indicates otherwise and is used synonymously with this term.

[0091] As used herein, "TMPRSS6" refers to a type II transmembrane serine protease (TTSP) gene or protein. TMPRSS6 is also known as matriptase-2, IRIDA (iron-refractory iron deficiency anemia), transmembrane protease serine 6, type II transmembrane serine protease 6, and membrane-bound mosaic serine protease matriptase-2. TMPRSS6 is a serine protease type II transmembrane protein approximately 899 amino acids in length. TMPRSS6 contains multiple domains, such as a short internal region, a transmembrane domain, a sea urchin sperm protein / enteropeptidase domain / agrin (SEA) domain, two complement factor / sea urchin growth factor / BMP domains (CUB), three LDL-R class a domains (LDLa), and a trypsin-like serine protease domain with a conserved His-Asp-Ser triad (HDS). The term "TMPRSS6" includes human TMPRSS6 (whose amino acid and nucleotide sequences can be found, for example, in GenBank accession number GI:56682967); mouse TMPRSS6 (whose amino acid and nucleotide sequences can be found, for example, in GenBank accession number GI:125656151); rat TMPRSS6 (whose amino acid and nucleotide sequences can be found, for example, in GenBank accession number GI:194474097); and rhesus monkey TMPRSS6 (whose amino acid and nucleotide sequences can be found, for example, in GenBank accession numbers XM_001085203.2 (GI:297260989) and XM_001085319.1 (GI:109094061)). Further examples of AGT mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, OMIM, and the websites of the Macaca genome project.

[0092] As used herein, the term "TMPRSS6" also refers to variations in the native DNA sequence of the TMPRSS6 gene, such as single nucleotide polymorphisms (SNPs) in the TMPRSS6 gene. Exemplary SNPs can be found in the dbSNP database available at www.ncbi.nlm.nih.gov / projects / SNP.

[0093] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of the TMPRSS6 gene, including the mRNA which is the product of RNA processing of the primary transcript.

[0094] As used herein, the term "strand containing a sequence" refers to an oligonucleotide containing a nucleotide strand represented by a sequence denoted using standard nucleotide nomenclature.

[0095] "G", "C", "A", and "U" each generally represent nucleotides containing the bases guanine, cytosine, adenine, and uracil, respectively. "T" and "dT" are used synonymously herein and refer to deoxyribonucleotides in which the nucleobase is thymine, e.g., deoxyribothymidine, 2'-deoxythymidine, or thymidine. However, it will be understood that the terms "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" may also refer to modified nucleotides, or surrogate replacement moieties, as further detailed below. Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without significantly altering the base pairing properties of oligonucleotides containing nucleotides having such replacement moieties. For example, without limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced, for example, by a nucleotide containing inosine in the nucleotide sequences of the present invention. Sequences containing such replacement moieties are embodiments of the present invention.

[0096] As used interchangeably herein, the terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interference agent” refer to an agent that contains RNA and mediates the targeted cleavage of an RNA transcript via the RNA-induced silencing complex (RISC) pathway, as the terms are defined herein. iRNA induces sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). iRNA modulates (e.g., inhibits) the expression of TMPRSS6 in cells, such as cells in a mammalian subject.

[0097] In one embodiment, the RNAi agent of the invention comprises single-stranded RNA that interacts with a target RNA sequence, such as a TMPRSS6 target mRNA sequence, to effect cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is broken down into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3’ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex to allow the complementary antisense strand to direct target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Here, in one aspect, the invention relates to single-stranded RNA (siRNA) that is generated in cells and promotes the formation of a RISC complex that results in silencing of the target gene, i.e., the TMPRSS6 gene. Accordingly, the term “siRNA” is also used herein to refer to the RNAi described above.

[0098] In another embodiment, the RNAi agent can be a single-stranded siRNA that is introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides in length and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as a single-stranded siRNA that is chemically modified by the methods described herein or as described in Lima et al., (2012) Cell 150;:883-894.

[0099] In yet another embodiment, the present invention provides a single-stranded antisense oligonucleotide molecule that targets TMPRSS6. The "single-stranded antisense oligonucleotide molecule" is complementary to a sequence in the target mRNA (i.e., TMPRSS6). The single-stranded antisense oligonucleotide molecule can stoichiometrically inhibit translation by base pairing to the mRNA and physically interfering with the translation machinery (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). Alternatively, the single-stranded antisense oligonucleotide molecule inhibits the target mRNA by hybridizing to the target and cleaving the target by RNaseH cleavage events. The single-stranded antisense oligonucleotide molecule is about 10 to about 30 nucleotides in length and can have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can comprise at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from any one of the antisense nucleotide sequences described herein, such as any one of the sequences shown in Tables 1, 2, 4, 5, 8, 10, and 12, or can bind to any of the target sites described herein. The single-stranded antisense oligonucleotide molecule can comprise modified RNA, DNA, or combinations thereof.

[0100] In another embodiment, the "iRNA" for use in the compositions, uses, and methods of the present invention is double-stranded RNA and is referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent", or "dsRNA". The term "dsRNA" refers to a ribonucleic acid molecule complex having a double-stranded structure comprising two anti-parallel and substantially complementary nucleic acid strands that are shown to have "sense" and "antisense" orientations with respect to the target RNA, i.e., the TMPRSS6 gene. In certain embodiments of the present invention, the double-stranded RNA (dsRNA) causes degradation of the target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0101] Generally, most of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Further, as used herein, an "RNAi agent" may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications in multiple nucleotides. Such modifications may include any type of modification disclosed herein or known in the art. Any such modification when used in an siRNA type molecule is encompassed by an "RNAi agent" for the purposes of this specification and the claims.

[0102] The two strands forming the double-stranded structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of one larger molecule and are thus joined by a continuous strand of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand, the joined RNA strands are referred to as a "hairpin loop". If the two strands are covalently joined by means other than a continuous strand of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand, the joined structure is referred to as a "linker". The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus the number of overhangs present in the double-stranded region. In addition to the double-stranded structure, an RNAi agent may include one or more nucleotide overhangs.

[0103] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, for example, a TMPRSS6 target mRNA sequence, to direct cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by type III endonucleases known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 base pair short interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to direct target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of an RNAi agent when the 3' end of one strand of the RNAi agent extends beyond the 5' end of the other strand, or vice versa. "Blunt" or "blunt end" means that there are no unpaired nucleotides at the corresponding end of the double-stranded RNAi agent, i.e., there is no nucleotide overhang. A "blunt end" RNAi agent is a dsRNA that is double-stranded over its entire length, i.e., there are 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 having one overhang and one blunt end) or RNAi agents having nucleotide overhangs at both ends.

[0104] The term "antisense strand" refers to the strand of a double-stranded RNAi agent (e.g., human TMPRSS6 mRNA) that contains a region substantially complementary to the target sequence. As used herein, the term "region complementary to a portion of the mRNA encoding transthyretin" refers to the region of the antisense strand that is substantially complementary to a portion of the TMPRSS6 mRNA sequence. When the complementary region is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions and, when present, generally occur within 6, 5, 4, 3, or 2 nucleotides of one or both of the terminal regions, e.g., the 5' and / or 3' ends.

[0105] As used herein, the term "sense strand" refers to the strand of a dsRNA that contains a region substantially complementary to the region of the antisense strand.

[0106] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site at which cleavage occurs in the target. In certain embodiments, the cleavage region comprises 3 bases immediately adjacent to the cleavage site at either end of the cleavage site. In certain embodiments, the cleavage region comprises 2 bases immediately adjacent to the cleavage site at either end of the cleavage site. In certain embodiments, the cleavage site specifically occurs at the site joined by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0107] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, as understood by one of ordinary skill in the art, refers to the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions to form a double-stranded structure. Such conditions can be, for example, stringent conditions, where stringent conditions can include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 12 to 16 hours at 50°C or 70°C, followed by washing. Other conditions can be applied, such as physiologically relevant conditions that can occur within an organism. For example, complementary sequences are sufficient for related functions of nucleic acids, such as to proceed RNAi. One of ordinary skill in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotides.

[0108] Arrays can be "perfectly complementary" to each other if there is base pairing between nucleotides containing a first nucleotide sequence and nucleotides containing a second nucleotide sequence over the entire length of the first and second nucleotide sequences. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be perfectly complementary or, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, upon performing hybridization, they may form one or more, but generally 4, 3 or 2 or fewer mismatched base pairs. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches with respect to the determination of complementarity. For example, for the purposes described herein, a dsRNA comprising one 21-nucleotide-long oligonucleotide and another 23-nucleotide-long oligonucleotide may be referred to as "perfectly complementary" if the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide.

[0109] As used herein, "complementary" sequences can include, or can be entirely formed from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobbles or Hoogsteen-type base pairs.

[0110] The terms "complementary", "perfectly complementary" and "substantially complementary" as used herein can be used in relation to matching bases between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood from the context of their use.

[0111] As used herein, a polynucleotide that is “substantially complementary to at least a portion of” messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of a target mRNA (e.g., an mRNA encoding TMPRSS6) that includes the 5′ UTR, open reading frame (ORF), or 3′ UTR. For example, a polynucleotide is complementary to at least a portion of TMPRSS6 mRNA if its sequence is substantially complementary to a contiguous portion of the mRNA encoding TMPRSS6.

[0112] As used herein, the term “inhibit” is used synonymously with “reduce,” “silence,” “downregulate,” “suppress,” and other similar terms, and includes inhibition at any level.

[0113] As used herein, the phrase “inhibit the expression of TMPRSS6” includes inhibition of the expression of any TMPRSS6 gene (e.g., the mouse TMPRSS6 gene, the rat TMPRSS6 gene, the monkey TMPRSS6 gene, or the human TMPRSS6 gene, etc.) as well as variants, (e.g., naturally occurring variants), or mutants of the TMPRSS6 gene. Thus, the TMPRSS6 gene can be a wild-type TMPRSS6 gene, a mutant TMPRSS6 gene, or a transgenic TMPRSS6 gene in the context of a genetically engineered cell, cell population, or organism.

[0114] "Inhibiting the expression of the TMPRSS6 gene" includes at least partial suppression of the expression of the TMPRSS6 gene, such as inhibition at any level of the TMPRSS6 gene, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0115] The expression of the TMPRSS6 gene can be evaluated based on the level of any variable related to the expression of the TMPRSS6 gene, such as the TMPRSS6 mRNA level, the TMPRSS6 protein level, the hepcidin mRNA level, the hepcidin protein level, or the serum lipid level in tissue or serum. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to the level of a control. The level of the control can be the level of any type of control used in the art, such as a pre-dose baseline level, or the level measured from a similar subject, cell, or sample treated with a non-treatment or control (e.g., a buffer-only control or an inert agent control).

[0116] As used herein, the phrase "contacting a cell with a double-stranded RNAi agent" includes contacting the cell by any possible means. The step of contacting a cell with a double-stranded RNAi agent includes the step of contacting the cell with the RNAi agent in vitro or the step of contacting the cell with the RNAi agent in vivo. The contact can be made directly or indirectly. Thus, for example, the RNAi agent may be physically contacted with the cell by performing the method individually, or the RNAi agent may be enabled to contact the cell later or placed in a situation where it will contact the cell later.

[0117] The step of contacting a cell in vitro can be performed, for example, by incubating the cell with the RNAi agent. The step of contacting a cell in vivo can be performed, for example, by injecting the RNAi agent into the tissue in which the cell is located or in the vicinity of the tissue, or by injecting the RNAi agent into another region, bloodstream or subcutaneous cavity, so that the RNAi agent will reach the tissue in which the cell is located later. For example, the RNAi agent may contain a ligand that directs the RNAi agent to the site of interest, such as the liver, such as a GalNAc3 ligand, and / or may be bound thereto. Combinations of in vitro and in vivo contact methods are also possible. In connection with the method of the present invention, the cell may also be contacted with the RNAi agent in vitro and then transplanted into a subject.

[0118] As used herein, "patient" or "subject" is intended to include either a human or a non-human animal, preferably a mammal, such as a human or a monkey. Most preferably, the subject or patient is a human.

[0119] As used herein, "diseases associated with TMPRSS6" is intended to include any disease that can be treated or prevented, or symptoms that can be alleviated, by inhibiting the expression of TMPRSS6. In certain embodiments, diseases associated with TMPRSS6 are also associated with iron overload, a condition characterized by elevated iron levels, or dysregulation of iron metabolism. Iron overload can be caused, for example, by genetic diseases, increased dietary iron intake, or parenteral administration of excess iron, including intravenous injection of excess iron, and transfusional hemosiderosis.

[0120] Diseases associated with TMPRSS6 include, but are not limited to, hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis, sideroblastic anemia, hemolytic anemia, myelodysplastic anemia, sickle cell anemia, abnormal hemoglobinopathy, thalassemia (e.g., β-thalassemia and α-thalassemia), chronic liver disease, late-onset cutaneous porphyria, erythropoietic porphyria, atransferrinemia, hereditary tyrosinemia, cerebrohepatorenal syndrome, idiopathic pulmonary hemosiderosis, and renal hemosiderosis.

[0121] Diseases associated with TMPRSS6 include diseases associated with oral administration of excess iron, transfusional hemosiderosis, and intravenous injection of excess iron.

[0122] Diseases associated with TMPRSS6 also include diseases having symptoms that may be associated with or caused by iron overload. Such symptoms include liver diseases (cirrhosis, cancer), heart attack or heart failure, diabetes, osteoarthritis, osteoporosis, metabolic syndrome, hypothyroidism, hypogonadism, and in some cases an increased risk of premature death. In one embodiment, diseases associated with TMPRSS6 include neurodegenerative diseases associated with iron overload and / or abnormal iron regulation, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Friedreich's ataxia, epilepsy, and multiple sclerosis. Administration of an iRNA targeting TMPRSS6, for example, an iRNA described in any one of Tables 1, 2, 4, 5, 8, 10, and 12, can treat one or more of these symptoms or prevent the onset or progression of a disease or disorder that is exacerbated by an increase in iron levels.

[0123] In one embodiment, a disease associated with TMPRSS6 is β-thalassemia. β-thalassemia is any one of a group of hereditary diseases characterized by a genetic defect in the synthesis of the β-globin chain. In the homozygous state, β-thalassemia ("severe thalassemia") causes severe, transfusion-dependent anemia. In the heterozygous state, the β-thalassemia trait ("mild thalassemia") causes mild to moderate microcytic anemia.

[0124] "Intermediate thalassemia" is a β-thalassemia that results in a subject whose clinical severity of the disease is around between the mild symptoms of mild β-thalassemia and severe β-thalassemia. The diagnosis is a clinical diagnosis based on patients who maintain a sufficient hemoglobin (Hb) value of at least 6 - 7 g / dL at the time of diagnosis without the need for regular transfusions.

[0125] In one embodiment, β-thalassemia is severe thalassemia. In another embodiment, β-thalassemia is intermediate thalassemia.

[0126] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a patient for treating a disease associated with TMPRSS6, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the manner of administration of the RNAi agent, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by TMPRSS6 expression, if any, type of previous treatment or co-treatment, and other individual characteristics of the patient being treated.

[0127] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject who has not yet developed or exhibited symptoms of a disease associated with TMPRSS6 but who is 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 includes delaying the progression of the disease or reducing the severity of a disease that develops later. A "prophylactically effective amount" can vary depending on the RNAi agent, the manner of administration of the agent, the risk of developing the disease, and the medical history, age, weight, family history, genetic makeup, if any, type of previous treatment or co-treatment, and other individual characteristics of the subject being treated.

[0128] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces a desired local or systemic effect that is a reasonable benefit / risk ratio applicable to any treatment. The iRNA agent (gent) used in the methods of the invention can be administered in an amount sufficient to obtain a reasonable benefit / risk ratio applicable to such treatment.

[0129] As used herein, the term "sample" includes similar body fluids, cells, or tissues isolated from a subject, as well as aggregates of body fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples derived from tissue, organs, or local regions. For example, a sample can be derived from a particular organ, a portion of an organ, or body fluid or cells within those organs. In certain embodiments, the sample can be derived from the liver (e.g., the whole liver or a particular portion of the liver or a particular type of cell in the liver such as a hepatocyte). In a preferred embodiment, "sample derived from a subject" refers to blood or plasma obtained from a subject. In a further embodiment, "sample derived from a subject" refers to liver tissue (or a subcomponent thereof) obtained from a subject.

[0130] II. iRNA of the Present Invention Disclosed herein are improved double-stranded RNAi agents that inhibit the expression of the TMPRSS6 gene in cells, such as cells in a mammal, e.g., a human, suffering from a disease associated with TMPRSS6, e.g., β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia) or hemochromatosis, and the use of such double-stranded RNAi agents.

[0131] Accordingly, the present invention provides double-stranded RNAi agents having chemical modifications capable of inhibiting the expression of a target gene (i.e., the TMPRSS6 gene) in vivo. In certain aspects of the invention, substantially all of the nucleotides of the iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of the iRNA of the invention are modified. The iRNA of the invention in which "substantially all of the nucleotides are modified" is mostly modified but not completely modified and can contain five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer unmodified nucleotides.

[0132] The RNAi agent includes a sense strand and an antisense strand. Each strand of the RNAi agent can be in the range of 12 to 30 nucleotides in length. For example, each strand can be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 19 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length.

[0133] The sense strand and the antisense strand typically form a double-stranded RNA (dsRNA), also referred to herein as an "RNAi agent". The double-stranded region of the RNAi agent can be 12 to 30 nucleotide pairs in length. For example, the double-stranded region can be 14 to 30 nucleotide pairs in length, 17 to 30 nucleotide pairs in length, 27 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 21 nucleotide pairs in length, 17 to 19 nucleotide pairs in length, 19 to 25 nucleotide pairs in length, 19 to 23 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, 21 to 25 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0134] In one embodiment, the RNAi agent may include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhang may be 1 to 6 nucleotides in length, for example, 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. The overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang may form a mismatch with the target mRNA, or the overhang may be complementary to the targeted gene sequence, or it may be another sequence. The first and second strands may also be joined by additional bases, for example, to form a hairpin, or by other non-base linkers.

[0135] In one embodiment, the nucleotides in the overhang region of the RNAi agent may each independently be modified or unmodified nucleotides including 2'-sugar modifications such as, but not limited to, 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT may be an overhang sequence for either end on either strand. The overhang may form a mismatch with the target mRNA, or the overhang may be complementary to the targeted gene sequence, or it may be another sequence.

[0136] The 5'- or 3'-overhangs in the sense strand, antisense strand or both strands of the RNAi agent can be phosphorylated. In certain embodiments, the overhang region comprises two nucleotides having phosphorothioates between two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'-overhang is present in the sense strand.

[0137] The RNAi agent can contain only one overhang that can enhance the interfering activity of RNAi without affecting its overall stability. For example, a single-stranded overhang can be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi can also have blunt ends located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt 5' end. Without wishing to be bound by theory, the asymmetric blunt ends at the 5' end of the antisense strand and the 3' end overhang of the antisense strand may advantageously act in the introduction of the guide strand into the RISC process.

[0138] Any of the nucleic acids employed in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated by reference. Modifications include, for example, terminal modifications, such as 5′-terminal modifications (phosphorylation, conjugation, inverted linkage) or 3′-terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.); base modifications, such as substitution of a base with a stable base, an unstable base, or a base that base pairs with a wide range of partners, removal of a base (non-basic nucleotide), or conjugated base; sugar modifications (e.g., at the 2′ or 4′ position) or substitution of the sugar; and / or backbone modifications including modification or substitution of the phosphodiester bond. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain a modified backbone or do not contain natural internucleoside linkages. RNAs having a modified backbone include, in particular, those that do not have a phosphorus atom in the 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 the internucleoside backbone can also be considered oligonucleosides. In certain embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0139] Examples of modified RNA backbones include phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methylphosphonate, and other alkylphosphonates including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-aminophosphoramidate and aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate having a normal 3'-5' linkage, their 2'-5' linkage analogs, and those having an inverted polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0140] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,195; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6,534,639; No. 6,608,035; No. 6,683,167; No. 6,858,715; No. 6,867,294; No. 6,878,805; No. 7,015,315; No. 7,041,816; No. 7,273,933; No. 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of which are hereby incorporated by reference into this specification.

[0141] Modified RNA backbones that do not contain phosphorus atoms within have a backbone formed by short-chain alkyl or cycloalkyl nucleoside internucleotide linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside internucleotide linkages, or one or more short-chain heteroatom or heterocyclic nucleoside internucleotide linkages. These include those having a morpholino linkage (partially formed from the sugar moiety of a nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having a combination of N, O, S, and CH 2 And others having component parts.

[0142] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 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, the entire contents of each of which are hereby incorporated by reference.

[0143] In other embodiments, suitable RNA mimics are contemplated for use in iRNA, where both the sugar and the internucleoside linkage, i.e., the backbone of the nucleotide unit, are replaced with novel groups. The base units are maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound that has been shown to have excellent hybridization properties is an RNA mimic called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, specifically, an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the azanitrogen atoms of the amide portions of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0144] One embodiment embraced by the present invention includes RNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly the --CH of U.S. Patent No. 5,489,677 described above. 2 --NH--CH 2 -、--CH 2 --N(CH 3 )--O--CH 2 --[known as the methylene(methylimino) or MMI backbone], --CH 2 --O--N(CH 3 )--CH 2 --、--CH 2 --N(CH 3 )--N(CH 3 )--CH 2 -- and --N(CH 3 )--CH 2 --CH 2 --[wherein the natural phosphodiester backbone is --O--P--O--CH 2--], and the amide backbone of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, the RNA featured herein has the morpholino backbone structure of the aforementioned U.S. Patent No. 5,034,506.

[0145] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein, may include one of the following at the 2' position: 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 are substituted or unsubstituted C 1 ~C 10 Alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include O[(CH 2 ) n O] m CH 3 , O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are from 1 to about 10. In other embodiments, the dsRNA comprises at the 2' position: 1 ~C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH3 、ONO 2 、NO 2 、N 3 、NH 2 、 heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group that improves the pharmacodynamic properties of iRNA, or a group that improves the pharmacokinetic properties of iRNA, and one of other substituents having similar properties. In certain embodiments, the modification is 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-MOE), i.e., 2'-O--CH 2 CH 2 OCH 3 )(Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., includes an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, described in the examples hereinbelow, i.e., O(CH 2 ) 2 ON(CH 3 ) 2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethylethyl or 2'-DMAEOE), i.e., 2'-O--CH 2 --O--CH 2 --N(CH 2 ) 2 .

[0146] Other modifications are 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of the iRNA, particularly at the 3' position of the 3' terminal nucleotide or the 3' position of the sugar in the 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. The iRNA can also have a sugar mimic such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are owned by the same owner as this application. The entire content of each of the above is hereby incorporated by reference into this specification.

[0147] iRNAs can also include nucleic acid base modifications or substitutions (often simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include deoxy-thymine (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, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methyladenine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine (daazaadenine) and 3-deazaguanine and 3-deazaadenine and other synthetic and natural nucleic acid bases.Additional nucleobases 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, J.L., 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, S.T. and Lebleu, B., Eds., CRC Press, 1993. Some of these nucleobases are particularly useful for enhancing the binding affinity of the oligomeric compounds taken up in the present invention. These include 5 - substituted pyrimidines, 6 - azapyrimidines, and N - 2, N - 6, and O - 6 substituted purines, including 2 - aminopropyladenine, 5 - propynyluracil, and 5 - propynylcytosine. The 5 - methylcytosine substituent has been shown to increase nucleic acid duplex stability by 0.6 - 1.2 °C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276 - 278), and is an exemplary base substitution, especially when combined with 2’ - O - methoxyethyl sugar modification.

[0148] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases as well as other modified nucleobases include, but are not limited to, the above-mentioned 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; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated by reference.

[0149] The RNA of the iRNA can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, which ribose moiety includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo conformational configuration. Adding locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O.R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0150] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are incorporated herein by reference.

[0151] Potentially stabilizing modifications to the ends of RNA molecules can 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-docosanoyl-uridine-3”-phosphate, inverted base dT (idT), and the like. The disclosure of this modification can be found in PCT Publication No. WO 2011 / 005861 pamphlet.

[0152] A. Modified iRNA Containing the Motif of the Present Invention In certain embodiments of the present invention, the double-stranded RNAi agents of the present invention include, for example, agents having chemical modifications disclosed in U.S. Provisional Patent Application No. 61 / 561,710, filed November 18, 2011, or PCT / US2012 / 065691, filed November 16, 2012, the entire contents of each of which are incorporated herein by reference.

[0153] As shown in this specification and U.S. Provisional Patent Application No. 61 / 561,710, better results are obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or the antisense strand of the RNAi agent, particularly at or near the cleavage site. In certain embodiments, the sense strand and the antisense strand of the RNAi agent may alternatively be fully modified. The introduction of these motifs disrupts the modification pattern of the sense strand and / or the antisense strand, if present. The RNAi agent may be optionally conjugated with a GalNAc derivative ligand, for example, on the sense strand. The resulting RNAi agent exhibits better gene silencing activity.

[0154] More specifically, it has surprisingly been found that when the sense strand and the antisense strand of the double-stranded RNAi agent are modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent, the gene silencing activity of the RNAi agent is significantly improved.

[0155] 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 in 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 in three consecutive nucleotides at positions 11, 12, and 13 from the 5'-end.

[0156] In another embodiment, the RNAi agent is a blunt-ended double strand 20 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0157] In yet another embodiment, the RNAi agent is a blunt-ended double strand 21 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0158] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand. The sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end. One end of the RNAi agent is blunt while the other end comprises a two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of the three nucleotides being overhang nucleotides and the third nucleotide being a paired nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages 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 the nucleotides that are part of the motif, are modified nucleotides. In one embodiment, each residue is independently modified, e.g., in an alternating motif, with 2'-O-methyl or 3'-fluoro. Optionally, the RNAi agent further comprises a ligand (preferably, GalNAc 3 ).

[0159] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a nucleotide length of at least 25 and 29 or less, and a second strand having a length of 30 nucleotides or less, comprising at least one motif of three 2'-O-methyl modifications 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, the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the second strand length such that when the RNAi agent is introduced into mammalian cells, it reduces the expression of the target gene, and the dicer cleavage of the RNAi agent preferentially yields an siRNA comprising the 3' end of the second strand, thereby reducing the expression of the target gene in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0160] In one embodiment, the sense strand of the RNAi agent comprises at least one motif of three identical modifications in three consecutive nucleotides, and one of the motifs is present at the cleavage site of the sense strand.

[0161] In one embodiment, the antisense strand of the RNAi agent can also comprise at least one motif of three identical modifications in three consecutive nucleotides, and one of the motifs is present at or near the cleavage site of the antisense strand.

[0162] In an RNAi agent having a double-stranded region 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically near the 10th, 11th, and 12th positions from the 5'-end. Thus, three identical modified motifs can start counting from the first nucleotide from the 5'-end of the antisense strand, or from the first paired nucleotide within the double-stranded region from the 5'-end of the antisense strand, and can be present at the 9th, 10th, 11th positions; 10th, 11th, 12th positions; 11th, 12th, 13th positions; 12th, 13th, 14th positions; or 13th, 14th, 15th positions of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5'-end.

[0163] The sense strand of the RNAi agent may contain at least one motif of three identical modifications in three consecutive nucleotides at the cleavage site of the strand; the antisense strand may have at least one motif of three identical modifications in three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be aligned such that one motif of three nucleotides in the sense strand and one motif of three nucleotides in the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand 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 overlap, or all three nucleotides may overlap.

[0164] In one embodiment, the sense strand of the RNAi agent may include two or more motifs of three identical modifications in three consecutive nucleotides. The first motif may be present at or near the cleavage site of the strand, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif present in another part of the strand away from the motif at or near the cleavage site of the same strand. The wing modification is adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemical structures of the motifs are different from each other. When the motifs are separated by one or more nucleotides, the chemical structures may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be present at one end with respect to the first motif at or near the cleavage site or on either side of the lead motif.

[0165] Similar to the sense strand, the antisense strand of the RNAi agent may also include two or more motifs of three identical modifications in three consecutive nucleotides, and at least one of the motifs is present at or near the cleavage site of the strand. This antisense strand may also include one or more wing modifications in a sequence similar to the wing modifications that may be present in the sense strand.

[0166] In one embodiment, the wing modification in the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0167] In another embodiment, the wing modification in the sense strand 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.

[0168] When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may be located at the same end of the double-stranded region and may have an overlap of one, two, or three nucleotides.

[0169] When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand are such that two modifications from one strand are each located at one end of the double-stranded region and have an overlap of one, two, or three nucleotides; two modifications from one strand are each located at the other end of the double-stranded region and have an overlap of one, two, or three nucleotides; and two modifications from one strand are located on each side of the lead motif and can be aligned to have an overlap of one, two, or three nucleotides in the double-stranded region.

[0170] In one embodiment, all nucleotides in the sense strand and the antisense strand of the RNAi agent, including nucleotides that are part of the motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more changes to one or both of the non-bridging phosphate oxygen and / or the bridging phosphate oxygen; changes to the ribose sugar component, such as the 2'-hydroxyl of the ribose sugar; large-scale substitution of the phosphate moiety with a "dephospho" linker; modification or substitution of the natural base; and substitution or modification of the ribose-phosphate backbone.

[0171] Since nucleic acids are polymers of subunits, many modifications, such as modifications to bases, or to the phosphate moiety, or to unbonded O of the phosphate moiety, are present at repeated positions within the nucleic acid. In some cases, the modification may be present at all positions of interest in the nucleic acid, but in many cases it is not. As an example, the modification may be present only at the 3’ or 5’ terminal positions, or only in the terminal region, such as at positions on the terminal nucleotides or only on the last 2, 3, 4, 5, or 10 nucleotides of the strand. The modification may be present in double-stranded regions, single-stranded regions, or both. The modification may be present only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at unbonded O positions may be present only at one or both ends, or only in the terminal region, such as at positions on the terminal nucleotides or only on the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may be present in double-stranded and single-stranded regions, particularly at the ends. The 5’ end or both ends may be phosphorylated.

[0172] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide surrogates in single-stranded overhangs, such as 5’ or 3’ overhangs, or both. For example, it may be desirable to include purine nucleotides in the overhang. In certain embodiments, all or some of the bases in the 3’ or 5’ overhang may be modified, for example, with the modifications described herein. Modifications can include, for example, use of modifications at the 2’ position of the ribose sugar by modifications known in the art, such as use of deoxyribonucleotides, 2’-deoxy-2’-fluoro (2’-F) or 2’-O-methyl modifications instead of ribose sugars of nucleobases, and modifications of the phosphate group, such as phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0173] In one embodiment, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The strand may contain two or more modifications. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0174] Typically, at least two different modifications are present in the sense strand and the antisense strand. Those two modifications can be 2'-O-methyl or 2'-fluoro modifications, or others.

[0175] In one embodiment, N a and / or N b contains an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, with each modification being present at alternating nucleotides of one strand. Alternating nucleotides can refer to every other nucleotide, every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB···", "AABBAABBAABB···", "AABAABAABAAB···", "AAABAAABAAAB···", "AAABBBAAABBB···", or "ABCABCABCABC···", etc.

[0176] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modification at every other nucleotide, may be the same, but each of the sense strand or the antisense strand can be selected from several possibilities of modifications within an alternating motif such as "ABABAB···", "ACACAC···", "BDBDBD···", or "CDCDCD···".

[0177] In one embodiment, the RNAi agent of the present invention includes a modification pattern of alternating motifs in the sense strand that is shifted relative to the modification pattern of alternating motifs in the antisense strand. This shift can be such that the modified groups of the nucleotides in the sense strand correspond to different modified groups of the nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in the dsRNA duplex, the alternating motifs in the sense strand may start from "ABABAB" from the 5' to 3' of the strand, and the alternating motifs in the antisense strand may start from "BABABA" from the 5' to 3' of the strand within the duplex region. As another example, the alternating motifs in the sense strand may start from "AABBAABB" from the 5' to 3' of the strand, and the alternating motifs in the antisense strand may start from "BBAABBAA" from the 5' to 3' of the strand within the duplex region, thereby resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0178] In one embodiment, the RNAi agent includes a pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification in the sense strand, and this pattern has a shift relative to the pattern of alternating motifs of 2'-O-methyl modification and 2'-F modification in the antisense strand, i.e., the 2'-O-methyl modified nucleotides in the sense strand form base pairs with the 2'-F modified nucleotides in the antisense strand, and vice versa. The 1st position of the sense strand may start with a 2'-F modification, and the 1st position of the antisense strand may start with a 2'-O-methyl modification.

[0179] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand and / or the antisense strand interrupts the initial modification pattern present in the sense strand and / or the antisense strand. This interruption of the modification pattern of the sense strand and / or the antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides unexpectedly enhances the gene silencing activity against the target gene.

[0180] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotides adjacent to the motif is a modification different from the modification of the motif. For example, a part of the sequence containing the motif is "···N a YYYN b ···", where "Y" represents the modification of the motif of three identical modifications in three consecutive nucleotides, and "N a " and "N b " represent the modifications of the nucleotides adjacent to the motif "YYY" that are different from the modification of Y, and N a and N b can be the same or different modifications. Alternatively, N a and / or N b may or may not be present if a wing modification is present.

[0181] The RNAi agent may further include at least one phosphorothioate or methylphosphonate internucleotide linkage. The modification of the phosphorothioate or methylphosphonate internucleotide linkage may be present in any nucleotide of the sense strand or the antisense strand or both strands at any position of the strand. For example, the modification of the internucleotide linkage may be present in all nucleotides in the sense strand and / or the antisense strand; the modification of each internucleotide linkage may be present in an alternating pattern in the sense strand and / or the antisense strand; or the sense strand or the antisense strand may include the modification of both internucleotide linkages in an alternating pattern. The alternating pattern of the modification of the internucleotide linkage in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of the modification of the internucleotide linkage in the sense strand may have a shift relative to the alternating pattern of the modification of the internucleotide linkage in the antisense strand.

[0182] In one embodiment, the RNAi includes a modification of phosphorothioate or methylphosphonate nucleotide linkages in the overhang region. For example, the overhang region may include two nucleotides having a phosphorothioate or methylphosphonate nucleotide linkage between the two nucleotides. The modification of the nucleotide linkage may also be formed to bind the overhang nucleotide to the terminal paired nucleotide within the double-stranded region. For example, at least 2, 3, 4, or all of the overhang nucleotides may be linked by phosphorothioate or methylphosphonate nucleotide linkages, and optionally, there may be additional phosphorothioate or methylphosphonate nucleotide linkages that bind the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate nucleotide linkages between the three terminal nucleotides, two of the three nucleotides are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. These three terminal nucleotides may be present 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.

[0183] In one embodiment, the two-nucleotide overhang is at the 3' end of the antisense strand, there are two phosphorothioate nucleotide linkages between the three terminal nucleotides, two of the three nucleotides are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. Optionally, the RNAi agent may further have two phosphorothioate nucleotide linkages between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0184] In one embodiment, the RNAi agent comprises a mismatch with the target, a mismatch within the double strand, or a combination thereof. The mismatch can occur in the overhang region or the double strand region. 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 pairing, and the simplest approach is to examine the pairs for each individual pair, although similar or analogous analyses can also 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-standard pairings (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.

[0185] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the double strand region from the 5’ end of the antisense strand independently selected from the group of A:U, G:U, I:C, and a mismatch pair for promoting dissociation of the antisense strand at the 5’ end of the double strand, e.g., a non-canonical or non-standard pairing or a pairing containing a universal base.

[0186] In one embodiment, the nucleotide at position 1 within the double strand 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 within the double strand region from the 5’ end of the antisense strand is an AU base pair. For example, the first base pair within the double strand region from the 5’ end of the antisense strand is an AU base pair.

[0187] In one embodiment, the sense strand sequence has the formula (I): 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’(I) (where: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides; each N b represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q each represent an overhang nucleotide; where Nb and Y do not have the same modification; XXX, YYY, and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides) can be represented by. Preferably, YYY is all 2'-F modified nucleotides.

[0188] In one embodiment, N a and / or N b contains an alternating pattern of modifications.

[0189] In one embodiment, the YYY motif is present at or near the cleavage site of the sense strand. For example, when the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the YYY motif can start counting from the first nucleotide from the 5' end; or optionally, starting from the 5' end, counting from the first paired nucleotide within the double-stranded region, it can be present at or near the cleavage site of the sense strand (for example: it can be present at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13).

[0190] In one embodiment, i is 1, j is 0, or i is 0, j is 1, or both i and j are 1. Thus, the sense strand can be represented by the following formula: 5'n p -Na -YYY-N b -ZZZ-N a -n q 3’(Ib); 5’n p -N a -XXX-N b -YYY-N a -n q 3’(Ic); or 5’n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3’(Id).

[0191] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0192] When the sense strand is represented as formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0193] When the sense strand is represented as formula (Id), each N b independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5 or 6. Each N a may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0194] Each of X, Y, and Z may be the same as or different from each other.

[0195] In other embodiments, i is 0, j is 0, and the sense strand can be represented by the following formula: 5’n p -N a -YYY-N a -n q 3’(Ia).

[0196] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0197] In one embodiment, the antisense strand sequence of RNAi is of 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) (wherein: k and l are each independently 0 or 1; p’ and q’ are each independently 0 to 6; each N a ’ independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides; each N b ’ independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ’ and n q ’ independently represent overhang nucleotides; here, N b ’ and Y’ do not have the same modification; X’X’X’, Y’Y’Y’ and Z’Z’Z’ each independently represent one motif of three identical modifications in a three consecutive nucleotide) It can be represented by

[0198] In one embodiment, N a ’ and / or N b ’ includes modification of an alternating pattern.

[0199] The Y’Y’Y’ motif is present at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the Y’Y’Y’ motif starts counting from the first nucleotide from the 5’ end; or optionally, starting from the 5’ end, from the first paired nucleotide within the double-stranded region, it can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand. Preferably, the Y’Y’Y’ motif is present at positions 11, 12, 13.

[0200] In one embodiment, the Y’Y’Y’ motif consists entirely of 2’-OMe modified nucleotides.

[0201] 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.

[0202] Therefore, the antisense strand can be represented by the following formula: 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N a ’-n p’ 3’ (IIb); 5’n q’ -N a ’-Y’Y’Y’-N b ’-X’X’X’-n p’ 3’ (IIc); or 5’n q’ -N a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N b ’-X’X’X’-N a ’-n p’3’(IId).

[0203] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0. Each N a ' independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0204] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0. Each N a ' independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0205] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0. Each N a ' independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6.

[0206] In other embodiments, k is 0, l is 0, and the antisense strand can be represented by the following formula: 5’n p’ -N a’ -Y’Y’Y’-N a’ -n q’ 3’(Ia).

[0207] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15, or 2 to 10.

[0208] Each of X’, Y’ and Z’ can be the same as or different from each other.

[0209] Each nucleotide of the sense strand and the antisense strand can be independently modified with LNA, HNA, CeNA, 2’-methoxyethyl, 2’-O-methyl, 2’-O-allyl, 2’-C-allyl, 2’-hydroxyl, or 2’-fluoro. For example, each nucleotide of the sense strand and the antisense strand can be independently modified with 2’-O-methyl or 2’-fluoro. Each of X, Y, Z, X’, Y’ and Z’ can in particular represent a 2’-O-methyl modification or a 2’-fluoro modification.

[0210] In one embodiment, for the sense strand of the RNAi agent, when the double-stranded region is 21 nucleotides, starting to count from the first nucleotide from the 5’ end; or optionally, starting to count from the 5’ end, from the first paired nucleotide within the double-stranded region, it may contain a YYY motif present at positions 9, 10 and 11 of the strand; Y represents a 2’-F modification. The sense strand may further contain an XXX motif or a ZZZ motif as a wing modification at the end opposite to the double-stranded region; XXX and ZZZ each independently represent a 2’-OMe modification or a 2’-F modification.

[0211] In one embodiment, for the antisense strand, starting to count from the first nucleotide from the 5’ end; or optionally, starting to count from the 5’ end, from the first paired nucleotide within the double-stranded region, it may contain a Y’Y’Y’ motif present at positions 11, 12 and 13 of the strand; Y’ represents a 2’-O-methyl modification. The antisense strand may further contain an X’X’X’ motif or a Z’Z’Z’ motif as a wing modification at the end opposite to the double-stranded region; X’X’X’ and Z’Z’Z’ each independently represent a 2’-OMe modification or a 2’-F modification.

[0212] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a double strand with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0213] Therefore, the RNAi agent for use in the method of the present invention may contain a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double strand has the formula (III): Sense: 5’n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3’ Antisense: 3’n p ’ -N a ’ -(X’X’X’) k -N b ’ -Y’Y’Y’-N b ’ -(Z’Z’Z’) l -N a ’ -n q ’ 5’ (III) (Wherein: i, j, k, and l are each independently 0 or 1; p, p’, q, and q’ are each independently 0 to 6; Each N a and N a ’ represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and 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; Here, each n, which may or may not exist p ’, n p , n q ’, and n q represents, independently, an overhang nucleotide; XXX, YYY, ZZZ, X’X’X’, Y’Y’Y’, and Z’Z’Z’ each independently represent a motif of one of three identical modifications on three consecutive nucleotides) is represented by

[0214] 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.

[0215] Exemplary combinations of the sense and antisense strands forming the RNAi double strand include the following formulas: 5’n p -N a -YYY-N a -n q 3’ 3’n p ’ -N a ’ -Y’Y’Y’-N a ’ n q ’ 5’ (IIIa) 5’n p -N a -YYY-N b -ZZZ-N a -n q 3’ 3’n p ’ -N a ’ -Y’Y’Y’-N b ’ -Z’Z’Z’-N a ’ n q’ 5' (IIIb) 5’n p -N a -XXX-N b -YYY-N a -n q 3’ 3’n p ’ -N a ’ -X’X’X’-N b ’ -Y’Y’Y’-N a ’ -n q ’ 5’ (IIIc) 5’n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3’ 3’n p ’ -N a ’ -X’X’X’-N b ’ -Y’Y’Y’-N b ’ -Z’Z’Z’-N a -n q ’ 5’ (IIId)

[0216] When the RNAi agent is represented by the formula (IIIa), each N a represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides independently.

[0217] When the RNAi agent is represented by the formula (IIIb), each N b represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides independently. Each N a represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides independently.

[0218] When the RNAi agent is represented by formula (IIIc), each N b , N b ’ independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0219] When the RNAi agent is represented by formula (IIId), each N b , N b ’ independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a , N a ’ independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. N a , N a ’, N b and N b ’ each independently contains modifications in an alternating pattern.

[0220] Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same as or different from each other.

[0221] When the RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y’ nucleotides. Alternatively, at least two of the Y nucleotides may form base pairs with the corresponding Y’ nucleotides; or all three of the Y nucleotides may all form base pairs with the corresponding Y’ nucleotides.

[0222] When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can form a base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z' nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z' nucleotides.

[0223] When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X' nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X' nucleotides.

[0224] In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and / or the modification on the X nucleotide is different from the modification on the X' nucleotide.

[0225] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0, and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond. In yet another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0, and at least one n p’ is linked 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. In another embodiment, when the RNAi agent is represented by formula (IIId), N a modification is a 2'-O-methyl or 2'-fluoro modification, and n p ’ > 0, and at least one n p ’ is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0226] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a modification is a 2'-O-methyl or 2'-fluoro modification, and n p ’ > 0, and at least one n p ’ is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0227] In one embodiment, the RNAi agent is a multimer comprising at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), and the double strands are linked by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites.

[0228] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more double-strands represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), and the double-strands are linked by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the double-strands can target the same gene or two different genes; or each of the double-strands can target the same gene at two different target sites.

[0229] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are bound to each other at one or both of the 5'-end and the 3'-end, and optionally conjugated to a ligand. Each of the RNAi agents can target the same gene or two different genes; or each of the RNAi agents can target the same gene at two different target sites.

[0230] Various publications describe multimeric RNAi agents that can be used in the methods of the present invention. Such publications include WO 2007 / 091269 pamphlet, US Patent No. 7,858,769 specification, WO 2010 / 141511 pamphlet, WO 2007 / 117686 pamphlet, WO 2009 / 014887 pamphlet and WO 2011 / 031520 pamphlet, the entire contents of each of which are incorporated herein by reference.

[0231] RNAi agents comprising the conjugation of one or more carbohydrate moieties to an RNAi agent can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is thus replaced is herein referred 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 a heterocyclic system, i.e., one or more ring atoms may be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier may be monocyclic or may contain two or more rings, such as fused rings. The cyclic carrier may be a completely saturated ring system or may contain one or more double bonds.

[0232] The ligand can be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points" and (ii) at least one "tethering attachment point". As used herein, a "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally a backbone, such as a phosphate or a modified phosphate, such as a sulfur-containing, suitable for and available for incorporation of the carrier into the backbone of a ribonucleic acid. A "tethering attachment point" (TAP) refers, in certain embodiments, to a constituent ring atom of the cyclic carrier that connects a selected moiety, such as a carbon atom or a heteroatom (different from the atom providing the backbone attachment point). This moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier often contains a functional group, such as an amino group, or generally provides a suitable bond for the incorporation or tethering of another chemical component, such as a ligand, to the constituent ring.

[0233] The RNAi agent may be conjugated to a ligand via a carrier, and this carrier may be a cyclic group or a cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the cyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0234] In certain embodiments, the RNAi agent for use in the methods of the invention is an agent selected from the group of agents listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12. In one embodiment, when the agent is an agent listed in Table 12, the agent may lack a terminal dT.

[0235] The present invention further includes a double-stranded RNAi agent comprising any one of the sequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12, which contains a 5' phosphate or a 5' phosphate mimetic on the antisense strand (see, for example, PCT Publication No. WO 2011 / 005860). Further, the present invention includes a double-stranded RNAi agent comprising any one of the sequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12, which contains a 2'-fluoro group instead of a 2'-OMe group at the 5' end of the sense strand.

[0236] These agents may further include a ligand.

[0237] In one embodiment, the agent is AD-60940 (sense strand: CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96; antisense strand: usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg).

[0238] A. Ligand The double-stranded RNA (dsRNA) agent of the present invention can optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, the antisense strand, or both strands at the 3'-end, 5'-end, or both ends. For example, the ligand can be conjugated to the sense strand. In a preferred embodiment, the ligand is conjugated to the 3'-end of the sense strand. In a preferred embodiment, the ligand is a GalNAc ligand. In a particularly preferred embodiment, the ligand is GalNAc 3 is: [Chemical formula]

[0239] In certain embodiments, a ligand, such as a GalNAc ligand, is attached to the 3'-end of the RNAi agent. In one embodiment, the RNAi agent has the following schematic diagram [Chemical formula] (wherein X is O or S), and is conjugated to a ligand, such as a GalNAc ligand. In one embodiment, X is O.

[0240] Various components can be attached to the RNAi agent of the present invention. Preferred moieties are ligands that are attached directly or indirectly, preferably by a covalent bond, via an intervening tether.

[0241] In preferred embodiments, the ligand alters the distribution, targeting, or lifespan of the molecule into which the ligand is incorporated. In preferred embodiments, the ligand provides an improved affinity for a selected target, such as a molecule, cell or cell type, compartment, receptor, such as a compartment of a cell or organ, tissue, organ, or region of the body, compared to a species in which such a ligand is absent. A ligand that provides an improved affinity for a selected target is also referred to as a targeting ligand.

[0242] Some ligands may have endosome-lytic properties. An endosome-lytic ligand promotes the lysis of endosomes and / or the transport of the compositions of the present invention, or components thereof, from endosomes to the cytoplasm of cells. The endosome-lytic ligand can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosome-lytic ligand adopts its active conformation at the pH of the endosome. The "active" conformation is the conformation in which the endosome-lytic ligand promotes the lysis of endosomes and / or the transport of the compositions of the present invention, or components thereof, from endosomes to the cytoplasm of cells. Exemplary endosome-lytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68). In one embodiment, the endosome-lytic component can include chemical groups (e.g., amino acids) that undergo a change in charge or protonation in response to a change in pH. The endosome-lytic component can be linear or branched.

[0243] The ligand can improve the properties of transport, hybridization, and specificity, and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotides, or polymer molecules comprising any combination of the monomers described herein and / or natural or modified ribonucleotides.

[0244] The ligand can generally include, for example, therapeutic modifiers for improving uptake; diagnostic compounds or reporter groups for monitoring distribution, for example; crosslinking agents; and moieties that confer nuclease resistance. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0245] The ligand can include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer), etc. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudo-peptide-polyamine, peptide-mimicking polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or α-helix peptide.

[0246] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., an antibody that binds to a specific cell type such as a lectin, glycoprotein, lipid, or protein, e.g., a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptide mimetic, or an aptamer.

[0247] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, aminos, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamines, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole complex, Eu3+ tetraaza macrocycle complex), dinitrophenyl, HRP, or AP.

[0248] A ligand can be a molecule having specific affinity for binding to a protein, such as a glycoprotein, or a peptide, such as a co-ligand, or an antibody, such as an antibody that binds to a specific cell type such as a cancer cell, an endothelial cell, or an osteocyte. The ligand may also include hormones and hormone receptors. The ligand may also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of 38MAP kinase, or an activator of NF-κB.

[0249] A ligand can be a substance, such as a drug, that can improve the uptake of an iRNA agent into a cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell, for example, by disrupting the cytoskeleton. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0250] A ligand can increase the uptake of an oligonucleotide into a cell, for example, by activating an inflammatory response. Exemplary ligands that can provide such an effect include tumor necrosis factor α (TNFα), interleukin-1β, or interferon γ.

[0251] In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds to a serum protein, such as human serum albumin (HSA). The HSA-binding ligand enables the distribution of the conjugate to target tissues of the body, such as non-renal target tissues. For example, the target tissue can be the liver, including hepatocytes of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the resistance of the conjugate to degradation, (b) increase the targeting or transport to target cells or cell membranes, and / or (c) modulate the binding to serum proteins, such as HSA.

[0252] The binding of the conjugate to the target tissue can be regulated, for example, controlled using a lipid-based ligand. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney and thus less likely to be removed from the body. A lipid or lipid-based ligand that binds more weakly to HSA can be used to target the conjugate to the kidney.

[0253] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, the lipid-based ligand binds to HSA with sufficient affinity such that the conjugate is preferably distributed to non-renal tissues. However, the affinity is preferably not so strong that the HSA-ligand binding cannot be reversed.

[0254] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA such that the conjugate is preferably distributed to the kidney. Other moieties that target renal cells can also be used instead of or in addition to the lipid-based ligand.

[0255] In another aspect, the ligand is a moiety that is taken up by target cells, such as proliferating cells, such as a vitamin. These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, such as folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. HAS, low density lipoprotein (LDL) and high density lipoprotein (HDL) are also included.

[0256] In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it can be modified including the use of peptidomimetics, enantiomers, non-peptides or pseudo-peptide bonds, and D-amino acids. The helical agent is preferably an α-helix agent, which preferably has lipophilic and hydrophobic phases.

[0257] The ligand can be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule that can be folded into a defined three-dimensional structure similar to that of a natural peptide. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) It may also be a target moiety. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules including peptides, oligonucleotides, and proteins across the cell membrane. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO: 13) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO: 14) have been found to be capable of functioning as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably, the peptide or peptidomimetic bound to the iRNA agent via the incorporated monomer units is a peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or an RGD mimetic. The peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moiety can have structural modifications such as to enhance stability or direct conformational properties. Any of the structural modifications described later can be used. The RGD peptide moiety can be used to target tumor cells such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). The RGD peptide can facilitate the targeting of the iRNA agent to tumors in various other tissues including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide facilitates the targeting of the iRNA agent to the kidney. The RGD peptide can be linear or cyclic and can be modified, for example, glycosylated or methylated, to facilitate targeting to a specific tissue. For example, the glycosylated RGD peptide can target the iRNA agent to α V β 3It can be delivered to tumor cells that express it (Haubner et al., Jour. Nucl. Med., 42:326 - 336, 2001). Peptides that target markers abundant in proliferating cells can be used. For example, RGD - containing peptides and peptidomimetics can target cancer cells, especially cells that exhibit integrin. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides containing D - amino acids, as well as synthetic RGD mimetics can be used. In addition to RGD, other moieties that target integrin ligands can be used. Generally, such ligands can be used to control proliferating cells and angiogenesis. Preferred conjugates of this type of ligand target PECAM - 1, VEGF, or other oncogenes, for example, the oncogenes described herein.

[0258] A "cell - penetrating peptide" can penetrate cells, such as microbial cells like bacterial or fungal cells, or mammalian cells like human cells. Peptides that penetrate microbial cells can be, for example, α - helix linear peptides (e.g., LL - 37 or Ceropin P1), peptides containing disulfide bonds (e.g., α - defensin, β - defensin, or bactenecin), or peptides containing only one or two dominant amino acids (e.g., PR - 39 or indolicidin). Cell - penetrating peptides can also contain a nuclear localization signal (NLS). For example, cell - penetrating peptides can be amphipathic peptides such as MPG, which are derived from the fusion peptide domain of HIV - 1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717 - 2724, 2003).

[0259] In one embodiment, the targeting peptide can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, bactofilin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, antibacterial peptide derived from the intestine of Megalopta genalis (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H 2 A peptide, Xenopus peptide, esculentinis-1, and caerin. Several factors are thought to preferably maintain the integrity of helix stability. For example, the maximum number of helix-stabilizing residues (e.g., leu, ala, or lys) are used, and the minimum number of helix-destabilizing residues (e.g., proline, or cyclic monomer units are used. Capping residues are considered (e.g., Gly is an exemplary N-capping residue, and / or C-terminal amidation can be used to provide additional H-bonds to stabilize the helix. Stability can be provided by the formation of salt bridges between residues with opposite charges separated only at positions i±3 or i±4. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamic acid or aspartic acid.

[0260] Peptide and peptidomimetic ligands include natural or modified peptides, such as D- or L-peptides; α, β, or γ-peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds, i.e., peptide bonds, substituted with one or more urea bonds, thiourea bonds, carbamate bonds, or sulfonylurea bonds; or ligands having cyclic peptides.

[0261] The targeting ligand can be any ligand capable of targeting a specific receptor. Examples are clusters of sugars such as folates, GalNAc, galactose, mannose, mannose-6P, GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. The targeting ligand also includes integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligand can also be based on nucleic acids, such as aptamers. The aptamer can be unmodified or can have any combination of the modifications disclosed herein.

[0262] Examples of endosome releasing agents include imidazole, poly or oligoimidazole, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers having masked or unmasked cationic or anionic charges, dendrimers having masked or unmasked cationic or anionic charges.

[0263] PK regulator represents a pharmacokinetic modulator. Examples of PK regulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK regulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides containing some phosphorothioate linkages are also known to bind to serum proteins, and thus short-chain oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, are also suitable for the present invention as ligands (e.g., as PK modulating ligands).

[0264] Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for the present invention as PK-regulating ligands.

[0265] Other ligand conjugates suitable for the present invention are described in U.S. Patent Application No. 10 / 916,185, filed Aug. 10, 2004; U.S. Patent Application No. 10 / 946,873, filed Sep. 21, 2004; U.S. Patent Application No. 10 / 833,934, filed Aug. 3, 2007; U.S. Patent Application No. 11 / 115,989, filed Apr. 27, 2005, and U.S. Patent Application No. 11 / 944,227, filed Nov. 21, 2007, and these documents are hereby incorporated by reference in their entirety for all purposes.

[0266] When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands may have the same properties while other ligands have different properties. For example, the ligands may have targeting properties, endosome-lysing activity, or PK-regulating properties. In a preferred embodiment, all the ligands have different properties.

[0267] The ligand can be attached to the oligonucleotide at various positions, such as the 3'-end, 5'-end, and / or internal positions. In a preferred embodiment, the ligand is attached to the oligonucleotide via an intervening tether, such as a carrier described herein. The ligand or the linking ligand can be present on the monomer when the monomer is incorporated into the growing chain. In certain embodiments, the ligand can be incorporated by attachment to the "precursor" monomer after the "precursor" monomer has been incorporated into the growing chain. For example, a monomer having an amino-terminal tether (i.e., to which the ligand is not attached), such as TAP-(CH 2 ) n NH 2can be incorporated into a growing oligonucleotide chain. In subsequent operations, i.e., after incorporating the precursor monomer into the chain, a ligand having an electrophilic group, e.g., a ligand having a pentafluorophenyl ester group or an aldehyde group, can then be attached to the precursor monomer by binding the electrophilic group of the ligand to the terminal electrophilic group of the tether of the precursor monomer.

[0268] In another example, a monomer having a chemical group suitable for participating in a click chemical reaction can be incorporated, e.g., into an azide- or alkyne-terminated tether / linker. In subsequent operations, i.e., after incorporating the precursor monomer into the chain, a ligand having a complementary chemical group, e.g., an alkyne or an azide, can be attached to the precursor monomer by binding the alkyne and azide together.

[0269] In the case of a double-stranded oligonucleotide, the ligand can be attached to one or both strands. In certain embodiments, the double-stranded iRNA agent comprises a ligand conjugated to the sense strand. In other embodiments, the double-stranded iRNA agent comprises a ligand conjugated to the antisense strand.

[0270] In certain embodiments, the ligand can be conjugated to the nucleobase, sugar moiety, or internucleoside linkage of a nucleic acid molecule. Conjugation to a purine nucleobase or a derivative thereof can occur at any position including intra - and extra - ring atoms. In certain embodiments, the 2 -, 6 -, 7 -, or 8 - position of the purine nucleobase is attached to the conjugate moiety. Conjugation to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In certain embodiments, the 2 -, 5 -, and 6 - positions of the pyrimidine nucleobase can be substituted with the conjugate moiety. Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached to the conjugate moiety include the 2’, 3’, and 5’ carbon atoms. The 1’ position can also be attached to the conjugate moiety, such as in an abasic residue. The internucleoside linkage can also have a conjugate moiety. In the case of a phosphorus - containing linkage (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be directly attached to the phosphorus atom or attached to an O, N, or S atom attached to the phosphorus atom. In the case of an amine - containing or amide - containing internucleoside linkage (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or an adjacent carbon atom.

[0271] Any suitable ligand in the field of RNA interference can be used, and the ligand is typically a carbohydrate, such as a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, or polysaccharide.

[0272] The linker for conjugating the ligand to the nucleic acid includes the linkers described above. For example, the ligand can be one or more GalNAc (N - acetylglucosamine) derivatives attached via a divalent or trivalent branched linker.

[0273] In one embodiment, the dsRNA of the present invention is conjugated to a divalent and trivalent branched linker having a structure represented by any of formulas (IV) - (VII):

Chemical formula

Chemical formula

[0274] Trivalent conjugate GalNAc derivatives such as those of formula (VII) are particularly useful for use with an RNAi agent to inhibit the expression of a target gene:

Chemical formula

[0275] Examples of suitable divalent and trivalent branched linking groups for conjugation to GalNAc derivatives include, but are not limited to, the following compounds:

Chemical formula

Chemical formula

Chemical formula

[0276] In other embodiments, the RNAi agent for use in the method of the present invention is AD-59743.

[0277] III. Delivery of the iRNA of the present invention Delivery of the iRNA agent of the present invention to cells, such as cells in a subject, e.g., a human subject (e.g., a subject in need of an iRNA agent, such as a subject suffering from a disorder associated with TMPRSS6, such as hemochromatosis), can be performed in several different ways. For example, delivery can be effected by contacting the cells with the iRNA of the present invention either in vitro or in vivo. In vivo delivery can also be effected directly by administering to the subject a composition comprising the iRNA, e.g., dsRNA. Alternatively, in vivo delivery can be effected indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are further described below.

[0278] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO 94 / 02595, which are hereby incorporated by reference in their entirety). In the case of in vivo delivery, factors to be considered for delivering iRNA molecules include, for example, the biological stability of the molecule being delivered, prevention of non-specific effects, and accumulation of the molecule being delivered in the target tissue. The non-specific effects of iRNA can be minimized by local administration, for example, by direct injection or implantation into tissue, or by locally administering the formulation. Local administration to the site of treatment maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be adversely affected by or degrade the agent, and can reduce the total dose of iRNA molecules administered. Several studies have shown the success of gene product knockdown when iRNA is administered locally. For example, intravitreal delivery of VEGF dsRNA by injection in rhesus 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 showed prevention of neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice decreased tumor volume (Pille, J et al. (2005) Mol. Ther. 11:267-274) and could extend the survival of tumor-bearing mice (Kim, WJ et al., (2006) Mol. Ther. 14:343-350; Li, S et al., (2007) Mol. Ther. 15:515-523).RNA interference has also shown success by local delivery to the central nervous system by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P. H. et al. (2005) Gene Ther. 12:59 - 66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G. T., et al. (2004) Neuroscience 129:521 - 528; Thakker, E. R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270 - 17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594 - 602) and to the lung by intranasal administration (Howard, K. A. et al., (2006) Mol. Ther. 14:476 - 484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677 - 10684; Bitko, V. et al., (2005) Nat. Med. 11:50 - 55). To systemically administer iRNA for the treatment of diseases, the RNA can be modified or delivered using a drug delivery system; both methods serve to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutical carrier enables targeting of the iRNA composition to the target tissue and can also avoid unwanted off - target effects. The iRNA molecule can be modified by chemical conjugation to a lipophilic group such as cholesterol, which improves cell uptake and prevents degradation. For example, iRNA against ApoB conjugated to a lipophilic cholesterol moiety was systemically administered to mice and achieved knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173 - 178). Conjugation of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J. O. et al., (2006) Nat. Biotechnol. 24:1005 - 1015).In alternative embodiments, the iRNA can be delivered using a drug delivery system such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate binding of the iRNA molecule (negatively charged) and also improve interaction with the negatively charged cell membrane, allowing for efficient uptake of the iRNA by the cell. Cationic lipids, dendrimers, or polymers can be bound to the iRNA or induced to form vesicles or micelles (see, for example, Kim SH.et al.,(2008)Journal of Controlled Release 129(2):107-116) that encapsulate the iRNA. Formation of vesicles or micelles further protects the iRNA from degradation when administered systemically. Methods for making and administering cationic iRNA complexes are within the capabilities of one of ordinary skill 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, which are hereby incorporated by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al (2003), supra; Verma, U.N., et al., (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S., et al., (2006) Nature 441:111-114), cardiolipin (Chien, P.Y., et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E., et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D.A., et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al., (1999) Pharm. Res. 16:1799-1804). In certain embodiments, the iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Patent No. 7,427,605, which is hereby incorporated by reference in its entirety.

[0279] A. The iRNA of the invention encoded by a vector iRNAs targeting the TMPRSS6 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A., et al., TIG. (1996), 12:5-10; International PCT Publication No. WO 00 / 22113 pamphlet by Skillern, A. et al., International PCT Publication No. WO 00 / 22114 pamphlet by Conrad, and U.S. Patent No. 6,054,299 to Conrad). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed to allow inheritance as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0280] One or more individual strands of the iRNA can be transcribed from a promoter in an expression vector. When two separate strands are expressed, for example, to produce dsRNA, two separate expression vectors can be co-introduced into the target cells (e.g., by transfection or infection). Alternatively, each individual strand of the dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence so as to have a stem-loop structure.

[0281] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for the expression of the iRNAs described herein can be produced using an expression vector compatible with eukaryotic cells, preferably an expression vector compatible with vertebrate cells. Expression vectors for eukaryotic cells are well known in the art and are available from many commercial sources. Such vectors typically provide convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of the iRNA expression vector can be by systemic delivery, for example, by intravenous or intramuscular administration, or by readministration to the patient after administration to target cells transplanted from the patient, or by any other means that allows introduction into the desired target cells.

[0282] The iRNA expression plasmid can be transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO (trademark)). Multiple lipid transfections for knockdown via iRNAs targeting different regions of the target RNA over a period of one week or more are also envisioned by the present invention. The success of vector introduction into host cells can be monitored using various known methods. For example, transient transfection can be shown using a reporter such as a fluorescent marker such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured by using a marker that confers resistance to a particular environmental factor (e.g., an antibiotic and a drug), such as hygromycin B resistance, to the transfected cells.

[0283] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors including, but not limited to, lentiviral vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors or poxvirus vectors such as fowlpox, e.g., canarypox or fowl pox; and (j) helper-dependent or attenuated adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors will either integrate into the genome of the cell or not. The construct may, if desired, contain viral sequences for transfection. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors. Constructs for the recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of iRNA in the target cell. Other aspects considered for vectors and constructs are described further below.

[0284] Vectors useful for the delivery of iRNA will contain regulatory elements (promoters, enhancers, etc.) sufficient for the expression of iRNA in the desired target cell or tissue. The regulatory elements can be selected to provide either constitutive expression or regulatable / inducible expression.

[0285] The expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as blood glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8: 20-24). Such inducible expression systems suitable for controlling the expression of dsRNA in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). One of ordinary skill in the art will be able to select an appropriate regulatory / promoter sequence based on the intended use of the iRNA transgene.

[0286] 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 proper packaging of the viral genome and integration into host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors that facilitate delivery of the nucleic acid to the patient. Further details regarding retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references demonstrating the use of retroviral vectors in gene therapy are Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors that may be considered for use include, for example, the HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0287] Adenoviruses are also contemplated for use in the delivery of the iRNAs of the present invention. Adenoviruses are, for example, particularly attractive vehicles for delivering genes to the respiratory epithelium. Adenoviruses are naturally infective to the respiratory epithelium and cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, the 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) presents an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of an adenovirus vector to transfer genes to the respiratory epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155; Mastrangeli et al. (1992), J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94 / 12649 pamphlet; and Wang et al., Gene Therapy 2:775-783 (1995). An AV vector suitable for expressing the iRNAs taken up by the present invention, a method for constructing a recombinant AV vector, and a method for delivering the vector into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0288] 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 can be expressed as two separate complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA taken up in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94 / 13788 pamphlet; and International Patent Application No. WO 93 / 24641 pamphlet, the entire disclosures of which are incorporated herein by reference.

[0289] Another viral vector suitable for delivery of the iRNA of the present invention is a poxvirus such as vaccinia virus, for example, attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or avipox such as fowlpox or canarypox.

[0290] The tropism of viral vectors can be modified by pseudotyping the vector with an envelope protein or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by manipulating the vector to express different capsid protein serotypes. See, for example, Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0291] The pharmaceutical formulation of the vector can contain the vector in an acceptable diluent or can contain a sustained-release matrix in which the gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical formulation can contain one or more cells that produce the gene delivery system.

[0292] IV. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations containing the iRNA of the present invention. In one embodiment, there is also provided herein a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition containing iRNA is useful for treating diseases or disorders associated with TMPRSS6, such as hemochromatosis. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic delivery via parenteral administration, such as intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, for example, by continuous pump infusion.

[0293] The pharmaceutical composition containing the RNAi agent of the present invention can be, for example, a solution with or without a buffer, or a composition containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micelle formulations, emulsions, and gene therapy vectors.

[0294] In the method of the present invention, the RNAi agent can be administered in solution. The free RNAi agent can be administered in a non-buffered solution, such as physiological saline or water. Alternatively, the free siRNA can also be administered in a suitable buffer. The buffer can contain acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffer is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer containing the RNAi agent can be adjusted to be suitable for administration to a subject.

[0295] In certain embodiments, the buffer further contains an agent for controlling the osmotic pressure of the solution such that the osmotic pressure is maintained at a desired value, such as the physiologic value of human plasma. Solutes that can be added to the buffer to control the osmotic pressure include, but are not limited to, proteins, peptides, amino acids, non-metabolic polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In certain embodiments, the agent for controlling the osmotic pressure of the solution is a salt. In a particular embodiment, the agent for controlling the osmotic pressure of the solution is sodium chloride or potassium chloride.

[0296] The pharmaceutical composition of the present invention can be administered in a dosage sufficient to inhibit the expression of the TMPRSS6 gene.

[0297] Generally, a preferred dosage of the iRNA of the present invention ranges from about 0.001 to about 200.0 milligrams per kilogram of the recipient's body weight per day, and generally ranges from about 1 to 50 mg per kilogram of body weight per day. For example, dsRNA can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single administration.

[0298] For example, an RNAi agent, such as dsRNA, can be administered at a dosage of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Intermediate values and ranges of the recited values are also intended to be part of the present invention.

[0299] In another embodiment, the RNAi agent, e.g., dsRNA, is from about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / mg, about 1.5 to about 45 mg / kb, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / mg, about 1.5 to about 40 mg / kb, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.Administered at a dose of 75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.1 to about 20 mg / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values intermediate to those recited and ranges are also intended to be part of the present invention.

[0300] For example, an RNAi agent, such as dsRNA, can be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values intermediate to those described and ranges are also intended to be part of the present invention.

[0301] In another embodiment, the RNAi agent, such as dsRNA, is from about 0.5 to about 50 mg / kg, from about 0.75 to about 50 mg / kg, from about 1 to about 50 mg / mg, from about 1.5 to about 50 mg / kg, from about 2 to about 50 mg / kg, from about 2.5 to about 50 mg / kg, from about 3 to about 50 mg / kg, from about 3.5 to about 50 mg / kg, from about 4 to about 50 mg / kg, from about 4.5 to about 50 mg / kg, from about 5 to about 50 mg / kg, from about 7.5 to about 50 mg / kg, from about 10 to about 50 mg / kg, from about 15 to about 50 mg / kg, from about 20 to about 50 mg / kg, from about 20 to about 50 mg / kg, from about 25 to about 50 mg / kg, from about 25 to about 50 mg / kg, from about 30 to about 50 mg / kg, from about 35 to about 50 mg / kg, from about 40 to about 50 mg / kg, from about 45 to about 50 mg / kg, from about 0.5 to about 45 mg / kg, from about 0.75 to about 45 mg / kg, from about 1 to about 45 mg / mg, from about 1.5 to about 45 mg / kb, from about 2 to about 45 mg / kg, from about 2.5 to about 45 mg / kg, from about 3 to about 45 mg / kg, from about 3.5 to about 45 mg / kg, from about 4 to about 45 mg / kg, from about 4.5 to about 45 mg / kg, from about 5 to about 45 mg / kg, from about 7.5 to about 45 mg / kg, from about 10 to about 45 mg / kg, from about 15 to about 45 mg / kg, from about 20 to about 45 mg / kg, from about 20 to about 45 mg / kg, from about 25 to about 45 mg / kg, from about 25 to about 45 mg / kg, from about 30 to about 45 mg / kg, from about 35 to about 45 mg / kg, from about 40 to about 45 mg / kg, from about 0.5 to about 40 mg / kg, from about 0.75 to about 40 mg / kg, from about 1 to about 40 mg / mg, from about 1.5 to about 40 mg / kb, from about 2 to about 40 mg / kg, from about 2.5 to about 40 mg / kg, from about 3 to about 40 mg / kg, from about 3.5 to about 40 mg / kg, from about 4 to about 40 mg / kg, from about 4.5 to about 40 mg / kg, from about 5 to about 40 mg / kg, from about 7.5 to about 40 mg / kg, from about 10 to about 40 mg / kg, from about 15 to about 40 mg / kg, from about 20 to about 40 mg / kg, from about 20 to about 40 mg / kg, from about 25 to about 40 mg / kg, from about 25 to about 40 mg / kg, from about 30 to about 40 mg / kg, from about 35 to about 40 mg / kg, from about 0.5 to about 30 mg / kg, from about 0.75 to about 30 mg / kg, from about 1 to about 30 mg / mg, from about 1.5 to about 30 mg / kb, from about 2 to about 30 mg / kg, from about 2.5 to about 30 mg / kg, from about 3 to about 30 mg / kg, from about 3.5 to about 30 mg / kg, from about 4 to about 30 mg / kg, from about 4.It is administered at a dose of 5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / mg, about 1.5 to about 20 mg / kb, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. In one embodiment, the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg. Intermediate values and ranges of the recited values are also intended to be part of the present invention.

[0302] For example, a therapeutic amount of iRNA such as about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg may be administered. Values intermediate to the recited values and ranges are also intended to be part of the present invention.

[0303] In certain embodiments, for example, when the composition of the present invention comprises the dsRNA and lipid described herein, a therapeutically effective amount of iRNA can be administered to a subject at about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 5 mg / kg, about 0.3 mg / kg to about 10 mg / kg, about 0.4 mg / kg to about 5 mg / kg, about 0.4 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg, etc. Values intermediate to the recited values and ranges are also intended to be part of the present invention.

[0304] For example, the dsRNA can be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Intermediate values and ranges of the values described are also intended to be part of the present invention.

[0305] In certain embodiments of the invention, for example, when a double-stranded RNAi agent comprises a modification (e.g., one or more motifs of three identical modifications on three consecutive nucleotides (including one such motif at or near the cleavage site of the agent)), six phosphorothioate linkages, and a ligand, such an agent is administered at about 0.01 to about 0.5 mg / kg, about 0.01 to about 0.4 mg / kg, about 0.01 to about 0.3 mg / kg, about 0.01 to about 0.2 mg / kg, about 0.01 to about 0.1 mg / kg, about 0.01 mg / kg to about 0.09 mg / kg, about 0.01 mg / kg to about 0.08 mg / kg, about 0.01 mg / kg to about 0.07 mg / kg, about 0.01 mg / kg to about 0.06 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.02 to about 0.5 mg / kg, about 0.02 to about 0.4 mg / kg, about 0.02 to about 0.3 mg / kg, about 0.02 to about 0.2 mg / kg, about 0.02 to about 0.1 mg / kg, about 0.02 mg / kg to about 0.09 mg / kg, about 0.02 mg / kg to about 0.08 mg / kg, about 0.02 mg / kg to about 0.07 mg / kg, about 0.02 mg / kg to about 0.06 mg / kg, about 0.02 mg / kg to about 0.05 mg / kg, about 0.03 to about 0.5 mg / kg, about 0.03 to about 0.4 mg / kg, about 0.03 to about 0.3 mg / kg, about 0.03 to about 0.2 mg / kg, about 0.03 to about 0.1 mg / kg, about 0.03 mg / kg to about 0.09 mg / kg, about 0.03 mg / kg to about 0.08 mg / kg, about 0.03 mg / kg to about 0.07 mg / kg, about 0.03 mg / kg to about 0.06 mg / kg, about 0.03 mg / kg to about 0.05 mg / kg, about 0.04 to about 0.5 mg / kg, about 0.04 to about 0.4 mg / kg, about 0.04 to about 0.3 mg / kg, about 0.04 to about 0.2 mg / kg, about 0.04 to about 0.1 mg / kg, about 0.04 mg / kg to about 0.09 mg / kg, about 0.04 mg / kg to about 0.08 mg / kg, about 0.04 mg / kg to about 0.07 mg / kg, about 0.04 mg / kg to about 0.06 mg / kg, about 0.05 to about 0.5 mg / kg, about 0.05 to about 0.4 mg / kg, about 0.05 to about 0.3 mg / kg, about 0.05 to about 0.2 mg / kg, about 0.05 to about 0.1 mg / kg, about 0.05 mg / kg to about 0.09 mg / kg, about 0.05 mg / kg to about 0.It is administered at a dose of 0.08 mg / kg, or about 0.05 mg / kg to about 0.07 mg / kg. Intermediate values and ranges of the values described above are also intended to be part of the present invention. For example, the RNAi agent can be administered to a subject at a dose of about 0.015 mg / kg to about 0.45 mg / mg.

[0306] For example, an RNAi agent, such as an RNAi agent in a pharmaceutical composition, can be administered at a dose of about 0.01 mg / kg, 0.0125 mg / kg, 0.015 mg / kg, 0.0175 mg / kg, 0.02 mg / kg, 0.0225 mg / kg, 0.025 mg / kg, 0.0275 mg / kg, 0.03 mg / kg, 0.0325 mg / kg, 0.035 mg / kg, 0.0375 mg / kg, 0.04 mg / kg, 0.0425 mg / kg, 0.045 mg / kg, 0.0475 mg / kg, 0.05 mg / kg, 0.0525 mg / kg, 0.055 mg / kg, 0.0575 mg / kg, 0.06 mg / kg, 0.0625 mg / kg, 0.065 mg / kg, 0.0675 mg / kg, 0.07 mg / kg, 0.0725 mg / kg, 0.075 mg / kg, 0.0775 mg / kg, 0.08 mg / kg, 0.0825 mg / kg, 0.085 mg / kg, 0.0875 mg / kg, 0.09 mg / kg, 0.0925 mg / kg, 0.095 mg / kg, 0.0975 mg / kg, 0.1 mg / kg, 0.125 mg / kg, 0.15 mg / kg, 0.175 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, or about 0.5 mg / kg. Intermediate values of the values described above are also intended to be part of the present invention.

[0307] The pharmaceutical composition can be administered once a day, or the iRNA can be administered as two, three or more sub-doses at appropriate intervals throughout the day, or even by delivery via continuous infusion or a sustained release formulation. In that case, the iRNA contained in each sub-dose needs to be correspondingly less in amount so as to achieve the total daily dose. The dosage unit may also be formulated for delivery over several days, for example using a conventional sustained release formulation that provides for the sustained release of the iRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering drugs to specific sites and can thus be used in conjunction with the drugs of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0308] In other embodiments, a single administration of the pharmaceutical composition can be long-lasting, such that subsequent doses are administered at intervals of 3, 4, or 5 days or less, or 1, 2, 3, or 4 weeks or less. In certain embodiments of the invention, a single administration of the pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single administration of the pharmaceutical composition of the invention is administered once every two months (bi-monthly).

[0309] One of ordinary skill in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for the individual iRNAs encompassed by the present invention can be approximated using conventional methodologies or based on in vivo testing using appropriate animal models as described elsewhere herein.

[0310] Advances in mouse genetics have generated many mouse models for the study of various human diseases, such as iron overload-related diseases that can benefit from reduced expression of TMPRSS6. Such models can be used for in vivo testing of iRNA and for determining effective dosages for treatment. Suitable mouse models are known in the art and include, for example, the thalassemia Th3 / + mouse as a model of β-thalassemia (Douet et al., Am. J. Pathol. (2011), 178(2):774-83), the HFE knockout mouse as a model of hereditary hemochromatosis (Zhou et al. (1998) Proc. Natl. Acad. Sci USA, 85:2492-2497); the Uros(mut248) mouse as a model of congenital erythropoietic porphyria (Ged et al. (2006) Genomics, 87(1):84-92).

[0311] The pharmaceutical composition of the present invention can be administered in several ways depending on whether local or systemic treatment is required and on the site to be treated. Administration can be local (e.g., by transdermal patch), pulmonary administration by inhalation or insufflation of a powder or aerosol, for example, by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous administration by, for example, an implant device; or intracranial administration by, for example, interstitial, intrathecal or intraventricular administration.

[0312] iRNA can be delivered to target specific tissues such as the liver (e.g., hepatocytes of the liver).

[0313] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, aerosols, solutions and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, etc. are required or may be desired. Coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the iRNA characterizing the present invention is a mixture with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoyl phosphatidylethanolamine DOPE, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), anionic (e.g., dimyristoyl phosphatidylglycerol DMPG) and cationic (e.g., dioleoyl tetramethylaminopropyl DOTAP and dioleoyl phosphatidylethanolamine DOTMA). The iRNA characterizing the present invention can be encapsulated in liposomes or can form complexes with liposomes, particularly cationic liposomes. Alternatively, the iRNA may be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides or pharmaceutically acceptable salts thereof). Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0314] A. iRNA Formulations Containing Membrane Molecular Assemblies The iRNAs for use in the compositions and methods of the present invention can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one bilayer or multiple bilayers. Liposomes include monolayer and multilayer vesicles having a membrane formed from lipophilic materials and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous exterior from the aqueous interior and usually does not contain the iRNA composition, but may in some cases. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Since the liposome membrane is structurally similar to the biological membrane, when the liposome adheres to the tissue, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the fusion of the liposome and the cell progresses, the internal aqueous contents containing the iRNA are delivered to the cell, where the iRNA can specifically bind to the target RNA and mediate RNAi. In some cases, the liposome is also specifically targeted, for example, to direct the iRNA to a specific cell type.

[0315] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent such that micelles are formed from the lipid component. For example, the lipid component can be an amphiphilic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Next, a preparation of the RNAi agent is added to the micelles containing the lipid component. The cationic groups in the lipid interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome formulation of the RNAi agent.

[0316] Optionally, a carrier compound that aids condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to assist condensation.

[0317] Methods for generating stable polynucleotide delivery vehicles incorporating polynucleotide / cationic lipid complexes as components of the delivery vehicle are further described, for example, in WO 96 / 37194 pamphlet, the entire content of which is incorporated herein by reference. Liposome formation can also include one or more aspects of the exemplary methods described in Felgner, P.L. et 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., M. Mol. Biol. 23:238, 1965; Olson et 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., Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication as well as freeze - thaw and extrusion (see, for example, Mayer et al., Biochim. Biophys. Acta 858:161, 1986). If consistently small (50 - 200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable for packaging preparations of RNAi agents into liposomes.

[0318] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are translocated into the endosome. The acidic pH within the endosome causes the liposomes to rupture and release their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0319] pH-sensitive and negatively charged liposomes do not complex with nucleic acids but rather entrap them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acids are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269~274).

[0320] One major type of liposome composition contains phospholipids other than naturally derived phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic membrane-fusion liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC) such as soy PC and egg PC, for example. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0321] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent No. 5,283,185; U.S. Patent No. 5,171,678; International Publication No. 94 / 00569 Pamphlet; International Publication No. 93 / 24640 Pamphlet; International Publication No. 91 / 16024 Pamphlet; 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.

[0322] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been tested and their usefulness 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) were used to deliver cyclosporin-A to the dermis of mouse skin. The results showed that such nonionic liposome systems are effective in promoting the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6)466).

[0323] Liposomes also include "sterically stabilized" liposomes, and as used herein this term refers to liposomes containing one or more specified lipids which, when incorporated into the liposome, result in an enhanced circulation lifetime compared to liposomes lacking such specified lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid moiety of the liposome is (A) monosialoganglioside G M1Those containing one or more glycolipids such as, or (B) those derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, in the art, with respect to sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes is thought to result from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0324] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactosylcerebroside sulfate, and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). Both U.S. Patent No. 4,837,028 to Allen et al. and International Publication No. 88 / 04924 pamphlet disclose liposomes containing (1) sphingomyelin and (2) ganglioside G M1 or galactosylcerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 pamphlet (Lim et al.).

[0325] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Non-cationic liposomes cannot fuse with cell membranes as efficiently, but can be taken up by macrophages in vivo and used to deliver RNAi agents to macrophages.

[0326] Further advantages of liposomes include the following: Liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; liposomes can protect RNAi agents encapsulated within their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0327] Small liposomes can be formed using N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a positively charged synthetic cationic lipid, which can spontaneously interact with nucleic acids to form lipid-nucleic acid complexes capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells and effecting delivery of RNAi agents (see, for example, Felgner, P. L. et al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987, and U.S. Patent No. 4,897,355 for a description of its use with DOTMA and DNA).

[0328] 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), a DOTMA analog, can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin (trademark; Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously adhere to the negatively charged cell surface, fuse with the cell membrane, and efficiently deliver functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonio)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are joined by ester rather than ether linkages.

[0329] Other reported cationic lipid compounds include those conjugated to one of two types of lipids and conjugated to various moieties including, for example, carboxyspermine, including compounds such as 5-carboxyspermidine dioctaoleylamide (“DOGS”) (Transfectam (trademark), Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermidine-amide (“DPPES”) (see, e.g., U.S. Patent No. 5,171,678).

[0330] Another cationic lipid conjugate involves derivatization of a lipid with cholesterol (``DC-Chol'') formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolyllysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0331] Liposomal formulations are particularly suitable for topical administration, and liposomes exhibit several advantages over other formulations. Such advantages include a reduction in side effects associated with a high systemic absorption rate of the administered drug, an increase in the accumulation of the administered drug at the desired target, and the ability to administer RNAi agents to the skin. In one implementation, liposomes are used to deliver RNAi agents to epidermal cells and also to facilitate the penetration of RNAi agents into dermal tissues, such as the skin. For example, liposomes can be applied topically. The topical delivery of drugs formulated as liposomes to the skin has been reported (see, for example, 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.J. and Fould-Fogerite, S., Biotechniques 6: 682-690, 1988; Itani, T. et al., Gene 56: 267-276, 1987; Nicolau, C. et al. (1987) Meth. Enz. 149: 157-176, 1987; Straubinger, R.M. and Papahadjopoulos, D. Meth. Enz. 101: 512-527, 1983; Wang, C.Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84: 7851-7855, 1987).

[0332] In addition, nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been investigated to determine their usefulness in the delivery of drugs to the skin. Nonionic liposomal formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs to the dermis of mouse skin. Such formulations containing RNAi agents are useful for treating skin diseases.

[0333] Liposomes containing iRNA can be made to be highly deformable. Such deformability can enable the liposomes to permeate pores smaller than the average radius of the liposomes. For example, transfersomes are a type of deformable liposome. Transfersomes can be made by adding a surface-activating factor, usually a surfactant, to a standard liposome composition. Transfersomes containing an RNAi agent can be delivered, for example, by subcutaneous infection, to deliver the RNAi agent to keratinocytes of the skin. To cross intact mammalian skin, the lipid vesicles must permeate a series of micropores each having a diameter of less than 50 nm under the influence of a suitable transdermal gradient. Further, due to their lipid properties, these transfersomes can be self-optimizing (e.g., adaptable to the shape of pores), self-healing, and in many cases reach their target without breaking, and in many cases can be self-loading.

[0334] Other formulations suitable for the present invention are described in U.S. Provisional Patent Application No. 61 / 018,616, filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed on January 2, 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed on March 26, 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed on April 22, 2008 and U.S. Provisional Patent Application No. 61 / 051,528, filed on May 8, 2008. The formulations suitable for the present invention are also described in PCT Application No. PCT / US2007 / 080331, filed on October 3, 2007.

[0335] Transferosomes are yet another type of liposome and are highly deformable lipid aggregates that are attractive candidates as drug delivery vehicles. Transferosomes can also be described as lipid droplets, which, due to their high deformability, can easily penetrate through pores smaller than the droplets. Transferosomes are adaptable to the environment in which they are used, e.g., self-optimizing (adapting to the shape of pores in the skin), self-healing, often reaching their target without much subdivision, and often self-loading. To prepare transferosomes, it is usually possible to add a surface edge activator, which is a surfactant, to a standard liposome composition. Transferosomes have been used to deliver serum albumin to the skin. Transferosome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0336] Surfactants have found wide use in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking a number of different types of surfactants, both natural and synthetic, is by use of the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0337] When the surfactant molecules are not ionized, this surfactant is classified as a nonionic surfactant. Nonionic surfactants find wide applications in pharmaceuticals and beauty products and can be used over a wide range of pH values. Generally, their HLB values range from 2 to about 18 depending on their structures. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Also included in this class are nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0338] When the surfactant molecules carry a negative charge when dissolved or dispersed in water, this surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acylamides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0339] When the surfactant molecules carry a positive charge when dissolved or dispersed in water, this surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0340] When the surfactant molecules have the ability to carry either a positive or a negative charge, this surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

[0341] The use of surfactants in pharmaceutical products, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0342] The iRNA for use in the methods of the present invention can also be provided as a micellar formulation. A "micelle" is defined herein as a particular type of molecular aggregate 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. When the environment is hydrophobic, the reverse arrangement exists.

[0343] A mixed micellar formulation suitable for delivery through a transdermal membrane can be prepared by mixing an aqueous solution of an siRNA composition, an alkali metal C 8 ~C 22 alkyl sulfate, and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polyidocanol alkyl ether and its analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles are formed by mixing substantially any kind of the components, but are formed by vigorous mixing to provide smaller-sized micelles.

[0344] In one method, a first micelle composition containing an siRNA composition and at least an alkali metal alkyl sulfate is prepared. Next, the first micelle composition is 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 of the micelle-forming compounds, and then adding the remaining micelle-forming compounds while mixing vigorously.

[0345] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added together with the micelle-forming components. An isotonic agent such as glycerin may also be added after the formation of the mixed micelle composition.

[0346] In the delivery of the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, and the dispenser is filled with a propellant. The propellant under pressure is in liquid form in the dispenser. The ratio of the components is adjusted so that the aqueous phase and the propellant phase become one, i.e., so that there is one phase. If two phases are present, for example, it is necessary to shake the dispenser before dosing a part of the contents by means of a metering valve. The dosing dose of the pharmaceutical is sprayed from the metering valve in a fine spray form.

[0347] The propellant may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0348] The specific concentration of the essential components can be determined by relatively simple experiments. For absorption through the oral cavity, it is often desirable to increase, for example, at least two-fold or three-fold the dosage for administration by injection or via the gastrointestinal tract.

[0349] B. Lipid Particles The iRNA, i.e., dsRNA, of the present invention may be completely encapsulated in a lipid formulation, such as in an LNP, or may form other nucleic acid-lipid particles.

[0350] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs typically include a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particles (e.g., a PEG-lipid conjugate). LNPs have an extended circulation lifetime after intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them extremely useful for systemic applications. LNPs include "pSPLP", which contains an encapsulated condensing agent-nucleic acid complex as shown in PCT Publication No. WO 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, the nucleic acid is resistant to nuclease degradation in an aqueous solution when present in the nucleic acid-lipid particles of the present invention. Nucleic acid-lipid particles, and methods for their preparation, 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 PCT Publication No. WO 96 / 40964.

[0351] In one embodiment, the lipid-to-drug ratio (mass / mass ratio) (e.g., the lipid-to-dsRNA ratio) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Intermediate ranges within the above ranges are also contemplated as part of the present invention.

[0352] 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-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoley (Dilinoley)oxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley (Dilinoley)oxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.(Cl), 1,2-dilinoleoyl-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyl-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) or an analog thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-bis((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-tetraene-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof may be used. The cationic lipid may comprise about 20 mol% to about 50 mol%, or about 40 mol% of the total lipids present in the particles.

[0353] 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 Oct. 23, 2008, which is incorporated herein by reference.

[0354] In one embodiment, the lipid-siRNA particles contain 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% cholesterol: 10% PEG-C-DOMG (mole percent) and have a particle size of 63.0 ± 20 nm and a siRNA / lipid ratio of 0.027.

[0355] The ionic / non-cationic lipid can be, without limitation, an anionic lipid or a neutral lipid including, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidy(phosphatidy)ethanolamine (SOPE), cholesterol, or a mixture thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0356] The conjugate lipid that inhibits aggregation of the particles can be, for example, polyethylene glycol (PEG)-lipid including, without limitation, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryl oxypropyl (Ci 2) PEG-dimyristyloxypropyl (Ci 4 ) PEG-dipalmityloxypropyl (Ci 6 ) or PEG-distearyloxypropyl (C 8 ). The conjugate lipid that prevents particle aggregation can be 0 mol% to about 20 mol%, or 2 mol% of the total lipids present in the particles.

[0357] In some embodiments, the nucleic acid-lipid particles further comprise, for example, about 10 mol% to about 60 mol% or about 48 mol% cholesterol of the total lipids present in the particles.

[0358] In one embodiment, lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed Mar. 26, 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). The respective stock solutions in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml, PEG-Ceramide C16, 100 mg / ml. Next, the ND98, cholesterol, and PEG-Ceramide C16 stock solutions can be combined, for example, in a molar ratio of 42:48:10. The combined lipid solution can be mixed with an aqueous dsRNA solution (e.g., in sodium acetate (pH 5)) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles are usually formed spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder such as 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 exchanged, for example, with phosphate buffered saline (PBS) at a pH of about 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

Chemical formula

[0359] The LNP01 formulation is described, for example, in International Publication No. WO 2008 / 042973 pamphlet, which is incorporated herein by reference.

[0360] Additional exemplary lipid-dsRNA formulations are set forth in Table A.

[0361] [Table 1]

[0362] [Table 2]

[0363] [Table 3]

[0364] DSPC: Distearoyl phosphatidylcholine DPPC: Dipalmitoyl phosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG having an average molecular weight of 2000) PEG-DSG: PEG-distearyl glycerol (C18-PEG, or PEG-C18) (PEG having an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG having an average molecular weight of 2000)

[0365] Formulations containing LNP (l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. WO 2009 / 127060, filed Apr. 15, 2009, which is incorporated herein by reference.

[0366] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366, filed January 29, 2009; U.S. Provisional Patent Application No. 61 / 156,851, filed March 2, 2009; U.S. Provisional Patent Application No., filed June 10, 2009; U.S. Provisional Patent Application No. 61 / 228,373, filed July 24, 2009; U.S. Provisional Patent Application No. 61 / 239,686, filed September 3, 2009, and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are hereby incorporated by reference in their entirety.

[0367] Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed June 10, 2010, the entire contents of which are hereby incorporated by reference.

[0368] Formulations containing ALNY-100 are described, for example, in International Patent Application No. PCT / US09 / 63933, filed November 10, 2009, which is hereby incorporated by reference in its entirety.

[0369] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009 and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are hereby incorporated by reference in their entirety.

[0370] Synthesis of Ionic / Cationic Lipids Any of the compounds used in the nucleic acid-lipid particles of the present invention, such as cationic lipids, can be prepared by known organic synthesis techniques including the methods described in detail in the examples. All substituents are as defined below unless otherwise indicated.

[0371] "Alkyl" means a linear or branched, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated linear alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc.; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, etc. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, etc.

[0372] "Alkenyl" means an alkyl as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative linear and branched alkenyls include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, etc.

[0373] "Alkynyl" means any alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative linear and branched alkynyls include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, etc.

[0374] "Acyl" means any alkyl, alkenyl, or alkynyl in which the carbon at the point of attachment is substituted by an oxo group as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0375] "Heterocyclic ring" is either saturated, unsaturated, or aromatic, and is a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic ring containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur (wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized), and this heterocyclic ring includes a bicyclic ring in which any of the above heterocyclic rings is condensed to a benzene ring. The heterocyclic ring can be bonded via any heteroatom or carbon atom. The heterocyclic ring includes heteroaryl as defined below. The heterocyclic ring includes morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, etc.

[0376] The terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl", and "optionally substituted heterocyclic ring", when substituted, mean that at least one hydrogen atom is substituted with a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are substituted. In this regard, the substituents include oxo, halogen, heterocyclic ring, -CN, -ORx, -NRxRy, -NRxC(=O)Ry, -NRxSO2Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SOnRx and -SOnNRxRy, wherein n is 0, 1, or 2, and Rx and Ry are the same or different and independently are hydrogen, alkyl, or heterocyclic ring, and each of the alkyl and heterocyclic ring substituents may be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -ORx, heterocyclic ring, -NRxRy, -NRxC(=O)Ry, -NRxSO2Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SOnRx and -SOnNRxRy.

[0377] "Halogen" means fluoro, chloro, bromo and iodo.

[0378] In certain embodiments, the methods of the present invention may require the use of protecting groups. Methods of protecting groups are well known to those skilled in the art (see, e.g., Protective Groups in Organic Synthesis, Green, T.W. et al., Wiley-Interscience, New York City, 1999). Briefly, a protecting group in the context of the present invention is any group that reduces or eliminates the unwanted reactivity of a functional group. A protecting group is added to a functional group to shield its reactivity during a particular reaction and is then removed, allowing the original functional group to reappear. In certain embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0379] Synthesis of Formula A In certain embodiments, the nucleic acid-lipid particles of the present invention have the formula A:

Chemical formula

[0380]

Chemical formula

[0381]

Chemical formula

[0382] Synthesis of MC3 The preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid and then successively with dilute aqueous sodium hydrogen carbonate solution. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed using a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1 - 5% methanol / dichloromethane elution gradient. The fractions containing the purified product were combined. The solvent was removed to give a colorless oil (0.54 g). Synthesis of ALNY-100

[0383] The synthesis of ketal 519 [ALNY-100] was carried out according to Scheme 3: [Chemical formula]

[0384] Synthesis of 515 In a two-necked round-bottom flask (1 L), a stirred suspension of LiAlH 4 (3.74 g, 0.09852 mol) in 200 ml of anhydrous THF was slowly added with a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF at 0 °C under a nitrogen atmosphere. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 hours. The progress of the reaction was monitored by TLC. After completion of the reaction (by TLC), the mixture was cooled to 0 °C and saturated Na 2 SO 4Quenched by careful addition of the solution. The reaction mixture was stirred at room temperature for 4 h and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. Volatiles were removed under reduced pressure to afford 515 hydrochloride as a white solid. Yield: 7.12 g 1 1H-NMR (DMSO, 400 MHz): δ = 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66 - 2.60 (m, 2H), 2.50 - 2.45 (m, 5H).

[0385] Synthesis of 516 To a stirred solution of compound 515 in 100 mL of dry DCM in a 250 mL two-necked round-bottom flask was added NEt 3 (37.2 mL, 0.2669 mol), and the mixture was cooled to 0 °C under a nitrogen atmosphere. N-(Benzyloxy-carbonyl-oxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM was added slowly, and then the reaction mixture was warmed to room temperature. After completion of the reaction (2 - 3 h by TLC), the mixture was washed successively with 1 N HCl solution (1 × 100 mL) and saturated NaHCO 3 solution (1 × 50 mL). Next, the organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated to give the crude material, which was purified by silica gel column chromatography to afford 516 as a sticky mass. Yield: 11 g (89%). 1 1H-NMR (CDCl 3 , 400 MHz): δ = 7.36 - 7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30 - 2.25 (m, 2H). LC-MS [M + H]+ 232.3 (96.94%).

[0386] Synthesis of 517A and 517B Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL of acetone and water (10:1) in a 500 mL one-neck round-bottom flask, to which N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added, followed by 4.2 mL of a 7.6% solution of OsO 4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (about 3 h), the mixture was quenched by the addition of solid Na 2 SO 3 and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO 3 (1 × 50 mL) solution, water (1 × 30 mL) and finally brine (1 × 50 mL). The organic phase was dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure. Purification of the crude material by silica gel column chromatography gave a mixture of diastereomers, which was separated by preparative HPLC. Yield: -6 g of crude 517A-peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ = 7.39~7.31 (m, 5H), 5.04 (s, 2H), 4.78~4.73 (m, 1H), 4.48~4.47 (d, 2H), 3.94~3.93 (m, 2H), 2.71 (s, 3H), 1.72~1.67 (m, 4H). LC-MS - [M+H] - 266.3, [M+NH4+] - 283.5 present, HPLC - 97.86%. Stereochemistry confirmed by X-ray.

[0387] Synthesis of 518 Compound 518 (1.2 g, 41%) was obtained as a colorless oil using the same procedure as described for the synthesis of compound 505. 1H-NMR (CDCl 3, 400 MHz): δ = 7.35~7.33 (m, 4H), 7.30~7.27 (m, 1H), 5.37~5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58~4.57 (m, 2H), 2.78~2.74 (m, 7H), 2.06~2.00 (m, 8H), 1.96~1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37~1.25 (br m, 36H), 0.87 (m, 6H). HPLC - 98.65%.

[0388] General procedure for the synthesis of compound 519 A solution of compound 518 (1 equiv) in hexane (15 mL) was added dropwise to an ice - cooled solution of LAH in THF (1 M, 2 equiv). After complete addition, the mixture was heated at 40 °C for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated Na 2 SO 4 aqueous solution, then filtered through celite and reduced to an oil. Pure 519 (1.3 g, 68%) was obtained by column chromatography, which was obtained as a colorless oil. 13 C NMR δ = 130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): C 44 H 80 NO 2 Calculated molecular weight (M + H)+ for 654.6, found 654.6.

[0389] Formulations prepared by either standard or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. The formulation should be a milky translucent solution free of aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles can be measured, for example, by light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). The particles should have a size of about 20 - 300 nm, for example 40 - 100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, and the percentage captured, are estimated using a dye exclusion assay. Samples of formulated dsRNA may be incubated with an RNA-binding dye such as Ribogreen (Molecular Probes) in the presence or absence of a formulation-disrupting surfactant, for example 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from samples containing surfactant against a standard curve. The percentage captured is determined by subtracting the "free" dsRNA content (measured by signal in the absence of surfactant) from the total dsRNA content. The percentage of captured dsRNA is typically >85%. In the case of LNP formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Suitable ranges are typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0390] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions, or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. A thickening agent, flavoring agent, diluent, emulsifying agent, dispersion aid or binder may be desired. In some embodiments, the oral formulation is one in which the dsRNA characterizing the present invention is administered with one or more permeation enhancers, surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxycholeno chenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucuronic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycolidihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of permeation enhancers, such as a fatty acid / salt combined with a bile acid / salt, is used. One exemplary combination is the sodium salts of lauric acid, capric acid and UDCA. Further permeation enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The DsRNA characterizing the present invention can be orally delivered in particulate form, including spray-dried particles or particles complexed to form micro- or nanoparticles.dsRNA complexing agents include poly - amino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkyl cyanoacrylates; cationized gelatin, albumin, starch, acrylate, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylates; DEAE - derivatized polyimines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N - trimethyl chitosan, poly - L - lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinyl pyridine, polythiodiethylaminomethyl ethylene P(TDAE), polyaminostyrene (e.g., p - amino), poly(methyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), poly(isohexyl cyanoacrylate), DEAE - methacrylate, DEAE - hexyl acrylate, DEAE - acrylamide, DEAE - albumin and DEAE - dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L - lactic acid), poly(DL - lactic acid - co - glycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations for dsRNA, and those formulations are described in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780 and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

[0391] Compositions and formulations for parenteral, intracerebral (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions, which can also include buffers, diluents, and other suitable additives such as, without limitation, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0392] The pharmaceutical compositions of the present invention include, without limitation, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components including, without limitation, pre-formed solutions, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases such as liver cancer, formulations that target the liver are particularly preferred.

[0393] The pharmaceutical formulations of the present invention, which can conveniently exist in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. Generally, the formulation is prepared by uniformly and intimately associating the active ingredient with a liquid carrier or a micronized solid carrier or both, and then shaping the product if necessary.

[0394] The compositions of the present invention can be formulated into any of a number of possible dosage forms, including, without limitation, 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, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0395] C. Further Formulations i. Emulsion The composition of the present invention can be prepared and formulated as an emulsion. An emulsion is typically a heterogeneous system in which one liquid is dispersed in another liquid, usually in the form of droplets having a diameter exceeding 0.1 μm (see, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). An emulsion is often a two-layer system containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, an emulsion can be of either the water-in-oil (w / o) or oil-in-water (o / w) type. When the aqueous phase is finely divided and dispersed as tiny droplets in a continuous oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as tiny droplets in a continuous aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.An emulsion can contain additional components in addition to the dispersed phase and the active drug, and these components can exist as a solution in the aqueous phase, the oil phase, or as a separate phase by themselves. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as required. Pharmaceutical emulsions can be multiple emulsions consisting of three or more phases, such as in the case of water-in-oil-in-water (o / w / o) and oil-in-water-in-oil (w / o / w) emulsions. Such composite formulations often provide certain advantages not offered by simple two-component emulsions. A multiple emulsion in which the individual oil droplets of an o / w emulsion enclose small water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets enclosed in small spheres of water stabilized in a continuous phase of oil provides an o / w / o emulsion.

[0396] Emulsions are characterized by having little or no thermodynamic stability. In many cases, the dispersed or discontinuous phase of the emulsion is well dispersed in the external or continuous phase and is maintained in this form by means of an emulsifier or through the viscosity of the formulation. Either phase of the emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Other means of stabilizing an emulsion include the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich, N.G., and Ansel, H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0397] Synthetic surfactants, also known as surface active agents, have found extensive applicability in the formulation of emulsions and are reviewed in the literature (see, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are generally amphiphilic and contain a hydrophilic portion and a hydrophobic portion. The ratio of the hydrophilicity to the hydrophobicity of a surfactant is referred to as the hydrophilic / lipophilic balance (HLB) and is a valuable means in the classification and selection of surfactants during the preparation of formulations. Surfactants can be classified into different types, namely, nonionic, anionic, cationic, and amphoteric, based on the nature of the hydrophilic group (see, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0398] Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases, such as anhydrous lanolin and hydrophilic petrolatum, take up water to form w / o emulsions, yet possess hydrophilic properties that maintain their semi-solid consistency. Micronized solids are used as good emulsifiers, particularly in combinations of surfactants and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0399] A very diverse group 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 esters, wetting agents, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0400] Hydrophilic colloids or hydrocolloids include natural rubbers and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These are dispersed in water or swell in water to form a colloidal solution that stabilizes the emulsion by forming a strong interfacial film around the droplets of the dispersed phase and by increasing the viscosity of the external phase.

[0401] Emulsions often contain a number of components such as carbohydrates, proteins, sterols, and phospholipids that can readily support the growth of microorganisms, so these formulations often incorporate preservatives. Commonly used preservatives included in the formulations are methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also usually added to emulsion formulations to prevent the deterioration of the formulations. Antioxidants used can be free radical scavengers such as tocopherol, alkyl gallates, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0402] The application of emulsion formulations via the skin, oral, and parenteral routes, as well as methods for their manufacture, 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, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery are very widely used because of their ease of formulation and effectiveness from the viewpoints 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, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral oil-based laxatives, fat-soluble vitamins, and high-fat nutritional formulations are included in materials that are commonly orally administered as o / w emulsions.

[0403] ii. Microemulsion In one embodiment of the present invention, the composition of iRNA and nucleic acid is formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and amphiphilic substances that is a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, a microemulsion is prepared by first dispersing oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an alcohol of intermediate chain length, to form a transparent system. Thus, a microemulsion has been described as a thermodynamically stable, isotropically transparent dispersion consisting of two immiscible liquids stabilized by an interfacial film of surfactant molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared using a combination of three to five components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is of the water-in-oil (w / o) type or the oil-in-water (o / w) type depends on the properties of the oil and surfactant used and on the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0404] Phenomenological approaches using state diagrams have been widely studied and provide those skilled in the art with extensive knowledge regarding methods of formulating microemulsions (see, for example, Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in formulations of thermodynamically stable droplets that form spontaneously.

[0405] Surfactants used in the preparation of microemulsions include, alone or in combination with co-surfactants, non-limitingly, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), decaglycerol decaoleate (DAO750). Usually, co-surfactants that are short-chain alcohols such as ethanol, 1-propanol, and 1-butanol penetrate into the surfactant film, and as a result, by forming an irregular film due to the void space generated between surfactant molecules, they play a role in increasing interfacial fluidity. However, microemulsions can be prepared without using co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, non-limitingly, water, an aqueous solution of a drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can include, non-limitingly, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolated glycerides, saturated polyglycolated C8-C10 glycerides, vegetable oils, and silicone oils.

[0406] Microemulsions are of particular interest from the perspective of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed for improving the oral bioavailability of drugs including peptides (see, for example, U.S. Patent No. 6,191,105, U.S. Patent No. 7,063,860, U.S. Patent No. 7,070,802, U.S. Patent No. 7,157,099, Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, protection of drugs from enzymatic hydrolysis, possible improvement in drug absorption due to changes in membrane fluidity and permeability induced by surfactants, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (see, for example, U.S. Patent No. 6,191,105, U.S. Patent No. 7,063,860, U.S. Patent No. 7,070,802, U.S. Patent No. 7,157,099, Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can form spontaneously when the components of the microemulsion are brought together at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides or iRNAs. Microemulsions are also effective for the transdermal delivery of active components in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to promote increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improved local cellular uptake of iRNAs and nucleic acids.

[0407] The microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and enhance the absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories - surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0408] iii. Particulates The RNAi agents of the present invention may be incorporated into particles, such as particulates. Particulates can be produced by spray drying, but may also be produced by other methods including freeze drying, evaporation, fluid bed drying, vacuum drying, or combinations of these techniques.

[0409] iv. Penetration Enhancers In one embodiment, the present invention uses various penetration enhancers to effect efficient delivery of nucleic acids, particularly iRNA, to the skin of an animal. Most drugs exist in solution in both ionized and non-ionized forms. However, generally only lipid-soluble or lipophilic drugs cross cell membranes readily. It has been found that non-lipophilic drugs can also cross cell membranes when the membrane to be crossed has been treated with a penetration enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the permeability of lipophilic drugs.

[0410] The penetration enhancer can be classified as belonging to one of five major categories, namely 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 above types of penetration enhancers is described in more detail below.

[0411] A surfactant (or "surface-active agent") is a chemical substance that, when dissolved in an aqueous solution, reduces the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of iRNA through the mucosa. 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, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0412] Examples of various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, their C 1~20 alkyl esters (e.g., methyl, isopropyl and t-butyl), as well as their monoglycerides and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, for example, Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0413] The physiological roles of bile include the promotion of 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 in: Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, as well as their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the natural components of bile as well as any of their synthetic derivatives.Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucuronic acid (glucholic acid) (sodium glucuronate), glycolic acid (sodium glycolate), glycochenodeoxycholic acid (sodium glycochenodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, 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 In: Remington’s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0414] Chelating agents used in connection with the present invention can be defined as compounds that remove metal ions from a solution by forming complexes with the metal ions, resulting in improved absorption of iRNA through the mucosa. Regarding the use of chelating agents as penetration enhancers in the present invention, most characterized DNA nucleases require divalent metal ions for catalysis and are thus inhibited by chelating agents, so the chelating agents have the further advantage of also acting as DNase inhibitors (Jarrett, J. Chromatogr., 1993, 618, 315 - 339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (see, 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).

[0415] As used herein, a penetration enhancer compound that is neither a chelator nor a surfactant exhibits slight activity as a chelating agent or surfactant, but can nevertheless be defined as a compound that promotes the absorption of iRNA through the gastrointestinal mucosa (see, for example, Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Examples of this type of penetration enhancer 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 non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0416] Agents that promote the uptake of iRNA at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids such as lipofectin (see U.S. Patent No. 5,705,188 to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (see WO 97 / 30731 pamphlet by Lollo et al.) are also known to promote the cellular uptake of dsRNA. Examples of commercially available transfection reagents include, in particular, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;(Madison, WI), TransFast™ 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; aExamples include D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec (trademark) / LipoGen (trademark) (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER (trademark) transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect (trademark) (B-Bridge International, Mountain View, CA, USA).

[0417] Penetration of the administered nucleic acid can be enhanced using other agents, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azone, and terpenes such as limonene and menthone.

[0418] v. carrier The predetermined composition of the present invention also incorporates a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can be inert (i.e., not having biological activity perse), but can, for example, be recognized as a nucleic acid or an analog thereof that reduces the bioavailability of a biologically active nucleic acid by an in vivo process such as degrading the biologically active nucleic acid or promoting the removal of nucleic acid from the circulation. Co-administration of the nucleic acid and the carrier compound, typically with an excess of the latter substance, can substantially reduce the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, perhaps due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of partial phosphorothioate in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0419] vi. Excipient In contrast to the 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 can be liquid or solid and are selected to provide the desired bulk, consistency, etc. when combined with the nucleic acid and other specified components of the pharmaceutical composition, taking into account the planned method of administration. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose, etc.); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate, etc.); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); tablet disintegrants (such as starch, sodium starch glycolate, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

[0420] Suitable organic or inorganic excipients that do not react detrimentally with the nucleic acid and are pharmaceutically acceptable for non-parenteral administration can also be used in formulating the compositions of the invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0421] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions, or nucleic acid solutions in liquid or solid oil bases in a normal solvent such as alcohol. The solutions can also contain buffers, diluents, and other suitable additives. Suitable organic or inorganic excipients that do not react detrimentally with the nucleic acid and are pharmaceutically acceptable for non-parenteral administration can be used.

[0422] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0423] vii. Other components The composition of the present invention may further contain other auxiliary components conventionally found in pharmaceutical compositions at their established usage levels in the art. Thus, for example, the composition can contain additional, compatible, pharmaceutically active materials such as, for example, anti-itch agents, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful for physically formulating various dosage forms of the composition of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifying agents, thickening agents and stabilizers. However, those materials need not unduly interfere with the biological activity of the components of the composition of the present invention when added. The formulation may be sterilized and, if desired, mixed with adjuvants that do not interact detrimentally with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts that affect osmotic pressure, buffers, coloring agents, flavoring agents and / or aromatic substances.

[0424] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain a stabilizer.

[0425] In certain embodiments, the pharmaceutical compositions contemplated by the present invention include (a) one or more iRNA compounds and (b) one or more agents that function by a non-RNAi mechanism and are useful for treating a bleeding disorder. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-obesity agents, anti-viral agents, and / or anti-fibrotic agents. Further, 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 protease inhibitors such as telbivudine, entecavir, and teraprevir, and other agents disclosed in, for example, U.S. Patent Application Publication Nos. 2005 / 0148548, 2004 / 0167116, and 2003 / 0144217 to Tung et al.; and U.S. Patent Application Publication No. 2004 / 0127488 to Hale et al.

[0426] The toxicity and treatment efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50 (lethal dose for 50% of the population) and ED 50 (dose therapeutically effective in 50% of the population). The dose ratio between the toxic and treatment effects is the therapeutic index and can be expressed as the LD 50 / ED 50 ratio. Compounds that exhibit a high therapeutic index are preferred.

[0427] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage amounts for use in humans. The dosage of the compositions contemplated herein in the present invention will generally be an ED 50It is within the range of blood concentration including []. The dosage can vary within this range depending on the dosage form used and the administration route used. For any compound used in the method taken up by the present invention, an effective dosage for treatment can be initially estimated from cell culture assays. The dosage is formulated to achieve, in an animal model, the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence, including the IC50 (i.e., the concentration of the test compound that achieves half of the maximum inhibition of symptoms), as measured in cell culture (e.g., to achieve a decrease in the concentration of the polypeptide). Such information can be used to more accurately determine a useful dosage in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0428] In addition to those administrations described above, the iRNA characterizing the present invention may be administered in combination with other known drugs effective for the treatment of pathological processes that can be mediated by iron overload and treated by inhibiting TMPRSS6 expression. In any case, the physician administering can adjust the amount and administration time of the iRNA using standard measures of effectiveness known in the art or described herein based on the observed results.

[0429] V. Method for inhibiting TMPRSS6 expression The present invention provides a method for inhibiting the expression of TMPRSS6 (matriptase-2) in cells. The method includes contacting the cells with an amount of an RNAi agent effective to inhibit the expression of TMPRSS6 in the cells, for example, a double-stranded RNAi agent, thereby inhibiting the expression of TMPRSS6 in the cells.

[0430] The step of contacting the cell with the double-stranded RNAi agent can be performed in vitro or in vivo. The step of contacting the cell with the RNAi agent in vivo includes the step of contacting a cell or cell population in a subject, such as a human subject, with the RNAi agent. Combinations of in vitro and in vivo contacting methods are also possible. The contacting can be done directly or indirectly as described above. Further, the step of contacting the cell can be done by a targeting ligand comprising any ligand described herein or known in the art. In a preferred embodiment, the targeting ligand is a carbohydrate moiety, such as GalNAc 3 a ligand, or any other ligand that directs the RNAi agent to a site of interest, such as the liver of a subject.

[0431] As used herein, the term "inhibit" is used synonymously with "reduce", "silence", "down-regulate" and other similar terms and includes inhibition at any level.

[0432] As used herein, the phrase "inhibit the expression of TMPRSS6" is intended to refer to the inhibition of the expression of any TMPRSS6 gene (e.g., mouse TMPRSS6 gene, rat TMPRSS6 gene, monkey TMPRSS6 gene, or human TMPRSS6 gene, etc.) as well as variants or mutants of the TMPRSS6 gene. Thus, the TMPRSS6 gene can be a wild-type TMPRSS6 gene, a mutant TMPRSS6 gene, or a transgenic TMPRSS6 gene in the context of a genetically engineered cell, cell population, or organism.

[0433] "Inhibiting the expression of the TMPRSS6 gene" includes inhibiting the TMPRSS6 gene at any level, for example, at least partial suppression of the expression of the TMPRSS6 gene. The expression of the TMPRSS6 gene can be evaluated based on the level of any variable associated with the expression of the TMPRSS6 gene, for example, the TMPRSS6 mRNA level, the TMPRSS6 protein level, or the lipid level, or a change in the level. This level can be evaluated, for example, in individual cells or cell populations, including samples derived from a subject.

[0434] Inhibition can be evaluated by a decrease in the absolute or relative level of one or more variables associated with TMPRSS6 expression compared to the level of a control. The level of the control can be the level of any type of control used in the art, for example, the pre-dose baseline level, or the level measured from a similar subject, cell, or sample treated with a non-treatment or a control (e.g., a buffer-only control or an inert agent control, etc.).

[0435] In certain embodiments of the methods of the invention, the expression of the TMPRSS6 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0436] Inhibition of TMPRSS6 gene expression is manifested as a decrease in the amount of mRNA expressed by a first cell or cell group (such cells may be present, for example, in a sample derived from a subject) that has been treated such that the TMPRSS6 gene is transcribed and its expression is inhibited (e.g., by contacting one or more cells with the RNAi agent of the present invention or by administering the RNAi agent of the present invention to a subject in which the cells are or were present), as compared to a second cell or cell group (control cells) that are substantially identical to the first cell or cell group except that they have not been so treated. In a preferred embodiment, the inhibition is evaluated by expressing the level of mRNA in the treated cells as a percentage of the level of mRNA in the control cells using the following formula:

Number

[0437] Alternatively, inhibition of TMPRSS6 gene expression can be evaluated in terms of a decrease in parameters functionally related to TMPRSS6 gene expression, such as TMPRSS6 protein expression, hepcidin gene or protein expression, or iron levels in tissues or serum. TMPRSS6 gene silencing can be measured in any cell expressing TMPRSS6, structurally or by genome engineering, and by any assay known in the art. The liver is the major site of TMPRSS6 expression. Other substantial sites of expression include the kidney and uterus.

[0438] Inhibition of TMPRSS6 protein expression is manifested as a decrease in the level of TMPRSS6 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived from a subject). As described above for the evaluation of mRNA suppression, inhibition of the protein expression level in the treated cell or cell group can be similarly expressed as a percentage of the level of protein in the control cell or cell group.

[0439] Control cells or cell populations that can be used to evaluate inhibition of TMPRSS6 gene expression include cells or cell populations that have not yet been contacted with the RNAi agent of the present invention. For example, control cells or cell populations are obtained from individual subjects (e.g., human or animal subjects) prior to treatment of the subject with the RNAi agent.

[0440] The level of TMPRSS6 mRNA expressed by a cell or cell population can be measured using any method known in the art for evaluating mRNA expression. In one embodiment, the level of expression of TMPRSS6 in a sample is measured by detecting the transcribed polynucleotide, or a portion thereof, e.g., the mRNA of the TMPRSS6 gene. RNA can be extracted from cells using RNA extraction techniques including, for example, acid phenol / guanidinium isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen) or PAXgene (PreAnalytix, Switzerland). Exemplary assay formats using ribonucleic acid hybridization include nuclear run-on assay, RT-PCR, RNase protection assay (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis.

[0441] In one embodiment, the level of expression of TMPRSS6 is measured using a nucleic acid probe. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific TMPRSS6. Probes can be synthesized by those skilled in the art or obtained from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, protein, antibody, and organic molecules.

[0442] The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe assays. One method for measuring mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to TMPRSS6 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by flowing the isolated mRNA over an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an Affymetrix gene chip array. Those skilled in the art can readily adapt known mRNA detection methods for use in measuring the level of TMPRSS6 mRNA.

[0443] Alternative methods for measuring the level of TMPRSS6 expression 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), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (U.S. Patent No. 5,854,033 to Lizardi et al.) or any other nucleic acid amplification method, for example, nucleic acid amplification and / or reverse transcription enzyme processes (for preparing cDNA) of mRNA in a sample, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when only a very small number of nucleic acid molecules are present. In certain embodiments of the invention, the level of TMPRSS6 expression is measured by quantitative fluorescent RT-PCR (i.e., TaqMan (trademark) System).

[0444] The expression level of TMPRSS6 mRNA can be monitored using membrane blots (such as those used in hybridization assays such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads or fibers (or any solid support containing the bound nucleic acid). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. Measurement of TMPRSS6 expression levels can also include the use of nucleic acid probes in solution.

[0445] In a preferred embodiment, the level of mRNA expression is evaluated using branched DNA (bDNA) assay or real-time PCR (qPCR). The use of these methods is described and exemplified in the examples provided herein.

[0446] The level of expression of the TMPRSS6 protein can be measured using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel-in precipitation reaction, absorbance spectroscopy, colorimetric analysis, spectrophotometric assay, flow cytometry, (simple or double) immunodiffusion, immunoelectrophoresis, Western blot, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, and the like.

[0447] As used herein, the term "sample" includes similar body fluids, cells, or tissues isolated from a subject, as well as aggregates of body fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, lymph fluid, urine, cerebrospinal fluid, saliva, ocular fluid, and the like. Tissue samples can include samples derived from tissues, organs or local regions. For example, a sample can be derived from a particular organ, a portion of an organ, or body fluids or cells within those organs. In certain embodiments, the sample can be derived from the liver (e.g., whole liver or a particular portion of the liver or a particular type of cell in the liver such as hepatocytes). In a preferred embodiment, "a sample derived from a subject" refers to blood or plasma obtained from a subject. In a further embodiment, "a sample derived from a subject" refers to liver tissue obtained from a subject.

[0448] In certain embodiments of the method of the invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. Inhibition of TMPRSS6 expression can be evaluated using measurement of the level or change in level of TMPRSS6 mRNA or TMPRSS6 protein in a sample derived from a body fluid or tissue from a specific site within the subject. In a preferred embodiment, the site is the liver. The site can also be a subsection or subgroup of cells from any one of the above sites. The site can also include cells that express a particular type of receptor.

[0449] VI. Methods of treating or preventing diseases associated with TMPRSS6 The present invention also provides methods of treating or preventing diseases and conditions that can be modulated by TMPRSS6 gene expression. For example, the compositions described herein can be used to treat diseases associated with iron overload, such as thalassemia (e.g., β-thalassemia or α-thalassemia), primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, erythropoietic porphyria, sideroblastic anemia, hemolytic anemia, myelodysplastic anemia, or sickle cell anemia. In one embodiment, the TMPRSS6 iRNA can be used to treat hemoglobinopathies. The TMPRSS6 iRNA of the present invention can also be used to treat elevated iron levels resulting from other conditions, such as chronic alcohol dependence.

[0450] In thalassemia, the bone marrow synthesizes an insufficient amount of hemoglobin chains; this in turn decreases red blood cell production and causes anemia. Either the α or β chain can be affected, but β-thalassemia is more common. Newborns are normal because the body still produces HbF, which does not have the β chain; during the first few months of life, the bone marrow switches to producing HbA and symptoms begin to appear.

[0451] β-thalassemia is caused by mutations in either the non-expression (β°) or low-expression (β+) alleles of the HBB gene, and β-thalassemia varies in severity according to genotype, including mild / β-thalassemia trait (β / β° or β / β+), intermediate β-thalassemia (β° / β+), and severe β-thalassemia (β° / β° or β”7β+).

[0452] Intermediate thalassemia (TI) typically shows little hemolysis, while severe β-thalassemia (TM) typically involves, for example, sufficient hemolysis to cause anemia and splenomegaly; and marked ineffective hematopoiesis causing bone marrow drive (skeletal changes, osteopenia), increased erythropoietin synthesis, hepatosplenomegaly, ingestion of hematopoietic agents (megaloblastic anemia), and hyperuricemia in the blood. The iRNAs of the present invention, for example, TMPRSS6 iRNA, are typically more suitable for treating iron overload associated with thalassemia such as TI (e.g., for treating solids having a β° / β+, β / β° or β / β+ genotype).

[0453] The symptoms of β-thalassemia also include iron overload that causes treatment-related complications such as endocrine disorders, liver fibrosis, and myocardial fibrosis. Administration of an iRNA agent targeting TMPRSS6 may be effective in treating one or more of these symptoms.

[0454] α-thalassemia is caused by mutations in either the non-expression (a°) or low-expression (a+) alleles of the HBA1 or HBA2 gene, or thalassemia varies in severity according to genotype, including thalassemia trait (-α / αα), Hb Bart type and hydrops fetalis (a° / a°), mild a-thalassemia (-- / αα), (-α / -α), and HbH disease (- / -a). When fewer a-globin chains are produced, excess β-chains in adults and excess γ-chains in newborns are brought about. Excess β-chains form unstable tetramers (hemoglobin H or HbH of 4β-chains) having an abnormal oxygen dissociation curve. Administration of an iRNA agent targeting TMPRSS6 may be effective in treating iron overload in a subject suffering from a-thalassemia.

[0455] Symptoms of hemochromatosis include, for example, abdominal pain, joint pain, fatigue, lack of energy, weakness, darkening of the skin (often referred to as "bronzing"), and loss of body hair. Administration of an iRNA agent targeting TMPRSS6 can be effective in treating one or more of these symptoms.

[0456] Other symptoms associated with iron overload include liver disease (cirrhosis, cancer), heart attack or heart failure, diabetes, osteoarthritis, osteoporosis, metabolic syndrome, hypothyroidism, hypogonadism, and in some cases an increased risk of premature death. Inappropriate use of iron leading to overload can also promote neurodegenerative diseases such as Alzheimer's disease, juvenile Parkinson's disease, Huntington's disease, epilepsy, and multiple sclerosis. Administration of an iRNA agent targeting TMPRSS6, such as the iRNAs described in Table 1 or 2, can treat one or more of these symptoms or prevent the onset or progression of diseases or disorders that are worsened by an increase in iron levels.

[0457] The method of the present invention further relates to the use of an iRNA agent or a pharmaceutical composition thereof, for example, in combination with other agents and / or other treatment methods, such as those currently used to treat these diseases, such as known agents and / or known treatment methods, for treating diseases associated with iron overload. For example, in certain embodiments, an iRNA agent targeting TMPRSS6 is administered, for example, in combination with an iron chelating agent (e.g., deferoxamine), folic acid, blood transfusion, phlebotomy, an agent for treating ulcers, an agent for increasing fetal hemoglobin levels (e.g., hydroxyurea), an agent for controlling inf...

Claims

1. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 by no more than 3 nucleotides, and the antisense strand comprising at least 15 contiguous nucleotides that differ from any one of the nucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 by no more than 3 nucleotides; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; A double-stranded RNAi agent, wherein the sense strand is conjugated to a ligand attached at the 3' end.

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

3. 2. The double-stranded RNAi agent of claim 1, wherein the sense strand and the antisense strand comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12.

4. 4. The double stranded RNAi agent of any one of claims 1 to 3, wherein at least one of the modified nucleotides is selected from the group consisting of a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-basic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5' phosphate or a 5' phosphate mimic, and a terminal nucleotide attached to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

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

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

7. 1. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 (matriptase-2) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding TMPRSS6, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; Each n may be present or absent. ｐ , n ｐ ', n ｑ , and n ｑ ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides; N ｂ the modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

8. The double-stranded RNAi agent of claim 7, wherein i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1.

9. 8. The double-stranded RNAi agent of claim 7, wherein k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1.

10. 8. The double-stranded RNAi agent of claim 7, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

11. The double-stranded RNAi agent of claim 7, wherein the YYY motif is at or near the cleavage site of the sense strand.

12. The double-stranded RNAi agent of claim 7 , wherein the Y′Y′Y′ motif is present at positions 11, 12, and 13 of the antisense strand at the 5′ end.

13. The double-stranded RNAi agent of claim 12, wherein said Y' is 2'-O-methyl.

14. Formula (III) is represented by formula (IIIa): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -Y'Y'Y'-N ａ’ -n ｑ’ 5'(IIIa) The double-stranded RNAi agent of claim 7, represented by:

15. Formula (III) is a compound represented by formula (IIIb): Sense: 5'n ｐ -N ａ -YYY-N ｂ -ZZZ-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -Y'Y'Y'-N ｂ’ -Z'Z'Z'-N ａ’ -n ｑ’ 5'(IIIb) (In the formula, each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides. The double-stranded RNAi agent of claim 7, represented by:

16. Formula (III) is represented by formula (IIIc): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -X'X'X'-N ｂ’ -Y'Y'Y'-N ａ’ -n ｑ’ 5'(IIIc) (In the formula, each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides. The double-stranded RNAi agent of claim 7, represented by:

17. Formula (III) represents the formula (IIId): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ｂ -ZZZ-N ａ -n ｑ 3' Antisense: 3'n ｐ’ -N ａ’ -X'X'X'-N ｂ’ -Y'Y'Y'-N ｂ’ -Z'Z'Z'-N ａ’ -n ｑ’ 5'(IIId) (In the formula, each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N ａ and N ａ ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides. The double-stranded RNAi agent of claim 7, represented by:

18. The double-stranded RNAi agent of claim 7, wherein the double-stranded region is 15 to 30 nucleotide pairs in length.

19. 20. The double-stranded RNAi agent of claim 18, wherein the double-stranded region is 17-23 nucleotide pairs in length.

20. 20. The double-stranded RNAi agent of claim 18, wherein the double-stranded region is 17-25 nucleotide pairs in length.

21. 20. The double-stranded RNAi agent of claim 18, wherein the double-stranded region is 23 to 27 nucleotide pairs in length.

22. 20. The double-stranded RNAi agent of claim 18, wherein the double-stranded region is 19-21 nucleotide pairs in length.

23. 20. The double-stranded RNAi agent of claim 18, wherein the double-stranded region is 21-23 nucleotide pairs in length.

24. The double-stranded RNAi agent of claim 7, wherein each strand has 15-30 nucleotides.

25. The double-stranded RNAi agent of claim 1 or 7, wherein each strand has 19-30 nucleotides.

26. 8. The double stranded RNAi agent of claim 7, wherein the modification on the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

27. 27. The double-stranded RNAi agent of claim 26, wherein the modification on the nucleotide is a 2'-O-methyl or a 2'-fluoro modification.

28. 10. The double-stranded RNAi agent of claim 1 or 7, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

29. The ligand is 【Chemistry 1】 The double-stranded RNAi agent according to claim 1 or 7,

30. The double-stranded RNAi agent of claim 1 or 7, wherein the ligand is attached to the 3' end of the sense strand.

31. Schematic diagram below 【Chemistry 2】 31. The double-stranded RNAi agent of claim 30, conjugated to the ligand of:

32. The double-stranded RNAi agent of claim 1 or 7, further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

33. 33. The double-stranded RNAi agent of claim 32, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand.

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

35. 34. The double stranded RNAi agent of Claim 33, wherein said strand is the sense strand.

36. 33. The double-stranded RNAi agent of claim 32, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand.

37. 37. The double-stranded RNAi agent of claim 36, wherein said strand is the antisense strand.

38. 37. The double stranded RNAi agent of claim 36, wherein said strand is the sense strand.

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

40. 40. The double stranded RNAi agent of claim 39, wherein said strand is the antisense strand.

41. The double-stranded RNAi agent of claim 32, wherein the RNAi agent comprises six to eight phosphorothioate internucleotide linkages.

42. The double-stranded RNAi of claim 41, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

43. The double-stranded RNAi agent of claim 1 or 7, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

44. The double-stranded RNAi agent of claim 7, wherein said Y nucleotide comprises a 2'-fluoro modification.

45. The double-stranded RNAi agent of claim 7, wherein the Y' nucleotide comprises a 2'-O-methyl modification.

46. The double-stranded RNAi agent of claim 7, wherein p'>0.

47. The double-stranded RNAi agent of claim 7, wherein p'=2.

48. 48. The double-stranded RNAi agent of claim 47, wherein q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA.

49. 48. The double-stranded RNAi agent of claim 47, wherein q'=0, p=0, q=0, and the p' overhanging nucleotides are non-complementary to the target mRNA.

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

51. At least one n ｐ 51. The double stranded RNAi agent of any one of claims 46-50, wherein: ' is linked to an adjacent nucleotide via a phosphorothioate bond.

52. All n ｐ 52. The double stranded RNAi agent of claim 51, wherein:

53. The double-stranded RNAi agent of claim 1 or 7, wherein the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12.

54. The double-stranded RNAi agent of claim 1 or 7, wherein the RNAi agent is selected from the group consisting of AD-59743, AD-60940, and AD-61002.

55. The double-stranded RNAi agent of claim 1 or 7, wherein the RNAi agent is AD-60940.

56. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 in a cell, comprising: the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end; A double-stranded RNAi agent, wherein the sense strand is conjugated at its 3' end to one or more GalNAc derivatives attached via a branched bivalent or trivalent linker.

57. 57. The double-stranded RNAi agent of claim 56, wherein all of said nucleotides of said sense strand and all of said nucleotides of said antisense strand comprise a modification.

58. 1. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 (matriptase-2) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding TMPRSS6, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; Each n may be present or absent. ｐ , n ｐ ', n ｑ , and n ｑ ' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

59. 1. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 (matriptase-2) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding TMPRSS6, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand. A double-stranded RNAi agent represented by:

60. 1. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 (matriptase-2) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding TMPRSS6, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; A double-stranded RNAi agent represented by:

61. 1. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 (matriptase-2) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding TMPRSS6, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) (In the formula: i, j, k, and l are each independently 0 or 1; Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; Each N ｂ and N ｂ ' independently represents an oligonucleotide sequence containing 0-10 nucleotides, either modified or unmodified, or combinations thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ the modification on Y is different from said modification on N ｂ the modification on Y' is different from said modification on Y'; the sense strand comprises at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; A double-stranded RNAi agent represented by:

62. 1. A double-stranded RNAi agent capable of inhibiting expression of TMPRSS6 (matriptase-2) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding TMPRSS6, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3'n ｐ '-N ａ '-Y'Y'Y'-N ａ '-n ｑ '5' (IIIa) (In the formula: Each n may be present or absent. ｐ , n ｑ , and n ｑ ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n ｐ '>0, and at least one n ｐ ' is linked to an adjacent nucleotide via a phosphorothioate bond; Each N ａ and N ａ ' independently represent oligonucleotide sequences containing 0-25 nucleotides, either modified or unmodified, or combinations thereof, each sequence containing nucleotides of at least two different modifications; YYY and Y'Y'Y' each independently represent one motif of three identical modifications on three consecutive nucleotides, said modifications being 2'-O-methyl or 2'-fluoro modifications; the sense strand comprises at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; A double-stranded RNAi agent represented by:

63. An RNAi agent selected from the group of RNAi agents listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12.

64. 53. The RNAi agent of claim 52, wherein the RNAi agent is selected from the group consisting of AD-59743, AD-60940, and AD-61002.

65. A composition comprising a modified antisense polynucleotide agent capable of inhibiting expression of TMPRSS6 in a cell, said agent comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 1, 2, 4, 5, 8, 10, and 12, and said polynucleotide is about 14 to about 30 nucleotides in length.

66. 65. A vector comprising the double-stranded RNAi agent of any one of claims 1, 7, 56, and 58-64.

67. A cell comprising the double-stranded RNAi agent of any one of claims 1, 7, 56, and 58-64.

68. A pharmaceutical composition comprising a double-stranded RNAi agent according to any one of claims 1, 7, 56 and 58 to 64, or a modified antisense polynucleotide agent according to claim 65, or a vector according to claim 66.

69. 69. The pharmaceutical composition of claim 68, wherein the RNAi agent is administered in a non-buffered solution.

70. 70. The pharmaceutical composition of claim 69, wherein the non-buffered solution is saline or water.

71. The pharmaceutical composition of claim 68, wherein the siRNA is administered with a buffer.

72. 72. The pharmaceutical composition of claim 71, wherein the buffer comprises an acetate buffer, a citrate buffer, a prolamine buffer, a carbonate buffer, or a phosphate buffer, or any combination thereof.

73. 73. The pharmaceutical composition of claim 72, wherein the buffer is phosphate buffered saline (PBS).

74. 1. A method for inhibiting TMPRSS6 expression in a cell, comprising: (a) contacting said cell with a double-stranded RNAi agent according to any one of claims 1, 7, 56, and 58-64, or a modified antisense polynucleotide agent according to claim 65, or a vector according to claim 66, or a pharmaceutical composition according to any one of claims 68-73; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the TMPRSS6 gene, thereby inhibiting expression of the TMPRSS6 gene in the cells.

75. 75. The method of claim 74, wherein the cell is in a subject.

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

77. 77. The method of any one of claims 74 to 76, wherein the TMPRSS6 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

78. 77. The method of any one of claims 74 to 76, wherein hepcidin gene expression is increased by at least about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, or about 5 fold.

79. 77. The method of any one of claims 74-76, wherein serum hepcidin concentration is increased by at least about 10%, about 25%, about 50%, about 100%, about 150%, about 200%, about 250%, or about 300%.

80. 77. The method of any one of claims 74 to 76, wherein serum iron concentration is reduced by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

81. 77. The method of any one of claims 74-76, wherein the percent transferrin saturation is reduced by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

82. A method for treating a subject suffering from a disease associated with TMPRSS6, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent according to any one of claims 1, 7, 56, and 58 to 64, or a modified antisense polynucleotide agent according to claim 65, or a vector according to claim 66, or a pharmaceutical composition according to any one of claims 68 to 73, thereby treating the subject.

83. 1. A method of treating a subject suffering from a disease associated with TMPRSS6, comprising subcutaneously administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, thereby treating the subject; the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; The sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end; substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end; The method, wherein the sense strand is conjugated at its 3' end to one or more GalNAc derivatives attached via a branched bivalent or trivalent linker.

84. 84. The method of claim 83, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

85. 84. The method of claim 82 or 83, wherein the subject is a human.

86. 86. The method of claim 85, wherein the human is afflicted with hereditary hemochromatosis, β-thalassemia, or erythroblastic porphyria.

87. 86. The method of claim 85, wherein the human is suffering from β-thalassemia.

88. 88. The method of claim 87, wherein the β-thalassemia is thalassemia major.

89. 88. The method of claim 87, wherein the β-thalassemia is thalassemia intermedia.

90. 86. The method of claim 85, wherein the human is suffering from a disease associated with iron overload.

91. 91. The method of claim 90, wherein the disease associated with iron overload is Parkinson's disease, Alzheimer's disease or Friedreich's ataxia.

92. 84. The method of claim 82 or 83, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 1 mg / kg to about 10 mg / kg.

93. 93. The method of claim 92, wherein the double-stranded RNAi agent is administered at a dose of about 0.1 mg / kg, about 1.0 mg / kg, or about 3.0 mg / kg.

94. 93. The method of claim 92, wherein the double-stranded RNAi agent is administered at a dose of about 1 mg / kg to about 10 mg / kg.

95. 93. The method of claim 92, wherein the double-stranded RNAi agent is administered subcutaneously.

96. 93. The method of claim 92, wherein the double-stranded RNAi agent is administered intravenously.

97. 93. The method of claim 92, wherein the RNAi agent is administered in two or more doses.

98. 98. The method of claim 97, wherein the RNAi agent is administered at an interval selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

99. 98. The method of claim 97, wherein the RNAi agent is administered once a week for up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5 weeks, or more.

100. 84. The method of claim 82 or 83, further comprising administering to the subject an iron chelator.

101. 101. The method of claim 100, wherein the iron chelator is selected from the group consisting of deferiprone, deferoxamine, and deferasirox.

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