TMPRSS6 iRNA COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

This invention relates to RNAi agents targeting the TMPRSS6 gene, such as double-stranded RNAi agents, and methods for inhibiting the expression of TMPRSS6 using such RNAi agents, as well as methods for treating subjects with TMPRSS6-related conditions, such as iron overload-related conditions, such as β-thalassemia or hemochromatosis.

Description

TMPRSS6 iRNA components and their usage [Related Applications] This application claims priority to 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 relates to U.S. Provisional Application No. 61 / 561,710, filed November 18, 2011, and PCT / US2012 / 065601, filed November 16, 2012. The entire contents of each of the foregoing applications are incorporated herein by reference. [Sequence List] This immediate application contains a sequence list, which was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 21, 2014, is named 121301-00720_SL.txt and is 449,620 bytes in size. This invention relates to RNAi agents targeting the TMPRSS6 gene, methods for inhibiting the expression of TMPRSS6 using such RNAi agents, and methods for treating subjects with TMPRSS6-related conditions. The TMPRSS6 (Transmembrane Protease, Serine 6) gene encodes TMPRSS6, also known as proteolytic enzyme-2, a type II serine protease. It is primarily found in the liver, but high levels of TMPRSS6 mRNA can also be detected in the kidneys, with lower levels detected in the uterus and even less in most other tissues (Ramsay et al., Haematologica (2009), 94(6), 840-849). TMPRSS6 plays a crucial role in iron homeostasis by binding to and degrading hepcidin activators and the BMP coreceptor HJV (hemojuvelin), thereby causing a decrease in hepcidin levels. The TMPRSS6 system consists of the following components: a short N-terminal intracytoplasmic tail, a type II transmembrane domain, a stem region composed of two extracellular CUB (complement factor Cls / Clr, urchin embryonic growth factor, and BMP (bone-forming protein)) domains, three LDLR (low-density lipoprotein receptor type A) domains, and a C-terminal trypsin-like serine protease domain. The extracellular domain contains a consensus site for N-glycosylation, and the intracytoplasmic tail region contains potential phosphorylation sites. Many conditions may be related to iron overload, characterized by elevated iron levels. Iron overload can lead to excessive iron deposition in various tissues, causing tissue and organ damage. Therefore, there is a need for effective treatments for conditions associated with iron overload. This invention provides compositions comprising RNAi agents, such as double-stranded iRNA agents, that target TMPRSS6. This invention also provides methods for inhibiting TMPRSS6 expression and treating TMPRSS6-related conditions, such as iron overload-related conditions, thalassemia, β-thalassemia, or hemochromatosis, using compositions of this invention. Accordingly, on one hand, the present invention provides an RNAi agent capable of inhibiting the intracellular expression of TMPRSS6 (protein lyase-2), such as a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 adjacent nucleotides, wherein no more than 3 nucleotides are different from any 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 adjacent nucleotides, wherein no more than 3 nucleotides are different from any 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. Wherein, all the nucleotides of the positive nucleotide and all the nucleotides of the antisense nucleotide are modified nucleotides, and The positive stock binds to a ligand linked to the 3'-terminus. In a single-state sample, all nucleotides of the positive-sense term and all nucleotides of the anti-sense term are modified nucleotides. In a single sample, the positive and negative sequences each contain a complementary region containing at least 15 adjacent nucleotides, wherein no more than 3 nucleotides are different from any of the antisense sequences listed in any of Tables 1, 2, 4, 5, 8, 10 and 12. In one state, at least one of the modified nucleotides is selected from the group consisting of: 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debased nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, N-morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, nucleotides containing 5'-phosphoate groups, nucleotides containing 5'-phosphates or 5'-phosphate variants (see, for example, PCT No. WO 2011 / 005860), and terminal nucleotides linked to cholesterol derivatives or dodecanoic acid didecylamine groups. In one state, at least one strand contains a 3' overhang of at least one nucleotide. In another state, at least one strand contains a 3' overhang of at least two nucleotides. On the other hand, the present invention provides an RNAi agent capable of inhibiting the intracellular expression of TMPRSS6 (protein lyase-2), such as a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a positive strand complementary to the antisense strand, wherein the antisense strand comprises a region complementary to a portion of the mRNA encoding TMPRSS6, wherein the length of each strand is approximately 14 to approximately 30 nucleotides, and wherein the double-stranded RNAi agent is represented by formula (III): Justice: 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' Antonym: 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: i, j, k, and l are each independently 0 or 1; p, p', q and q' are each independently represented by 0 to 6; N a With N a Each 'represents an oligonucleotide sequence comprising 0 to 25 nucleotides, wherein the nucleotides are modified or unmodified or a combination thereof, and each sequence comprises at least two differently modified nucleotides; N b With N b Each 'represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified or unmodified or a combination thereof; n p n p '、n q and n q Each of these may or may not exist, and each independently represents a drooping nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' are each independently represented by a motif consisting of three consecutive nucleotides modified in three identical ways. N b The modifier above is different from the modifier above Y, and N b The modifier above 'Y' is different from the modifier above 'Y'; and The positive ligand binds to at least one ligand. In one sample, 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 sample, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1. In a single-state sample, the XXX system is complementary to X'X'X', the YYY system is complementary to Y'Y'Y', and the ZZZ system is complementary to Z'Z'Z'. In a single-state sample, the YYY model system appears at or near the cleavage site of the positive stock. In a single-state sample, the Y'Y'Y' model system appears at positions 11, 12, and 13 of the 5'-end of the antisense. In the first state sample, Y' is a 2'-O-methyl group. In a single-state sample, equation (III) is expressed by equation (IIIa): Justice: 5' n p -N a -YY YN a -n q 3' Antonym: 3' n p' -N a' -Y'Y'Y'-N a' -n q' 5' (IIIa). In another state, equation (III) is expressed by equation (IIIb): Justice: 5' n p -N a -YY YN b -ZZ ZN a -n q 3' Antonym: 3' n p' -N a' -Y'Y'Y'-N b '-Z'Z'Z'-N a' -n q'5' (IIIb) Where, N b With N b Each 'series independently represents an oligonucleotide sequence containing one to five modified nucleotides. In the next state, equation (III) is expressed by equation (IIIc): Justice: 5' n p -N a -XX XN b -YY YN a -n q 3' Antonym: 3' n p' -N a' -X'X'X'-N b' -Y'Y'Y'-N a' -n q' 5' (IIIc) Where, N b With N b Each 'series independently represents an oligonucleotide sequence containing one to five modified nucleotides. In a single-state sample, equation (III) is expressed by equation (IIId): Justice: 5' n p -N a -XX XN b -YY YN b -ZZ ZN a -n q 3' Antonym: 3' n p' -N a' -X'X'X'-N b' -Y'Y'Y'-N b' -Z'Z'Z'-N a' -n q' 5' (IIId) Where, N b With N b Each 'represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and N a With N a Each 'series independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides. In one state, the double-stranded region is 15 to 30 nucleotide pairs long. In another state, the double-stranded region is 17 to 23 nucleotide pairs long. In yet another state, the double-stranded region is 17 to 25 nucleotide pairs long. In one state, the double-stranded region is 23 to 27 nucleotide pairs long. In another state, the double-stranded region is 19 to 21 nucleotide pairs long. In yet another state, the double-stranded region is 21 to 23 nucleotide pairs long. In one state, each strand has 15 to 30 nucleotides. In another state, each strand has 19 to 30 nucleotides. In one state, the modifications on the nucleotides are selected from the group consisting of: LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluorine, 2'-deoxy, 2'-hydroxy, and combinations thereof. In another state, the modifications on the nucleotides are 2'-O-methyl or 2'-fluorine modifications. In one state, the ligand system comprises one or more GalNAc derivatives linked by divalent and trivalent branched chain linkers. In another state, the ligand system... In a single state, the allocation system is connected to the 3' end of the justice stock. In a single-state sample, the RNAi system binds to the ligand as shown in the figure below. Where X is O or S. In a specific state sample, X is O. In one state, the agent system further comprises at least one internucleotide linkage of a thiophosphate or methylphosphonate. In one state, the internucleotide linkage of the thiophosphate or methylphosphonate is located at the 3'-terminus of one strand. In one state, the strand is an antisense strand. In another state, the strand is a right strand. In one state, the internucleotide linkage of the thiophosphate or methylphosphonate is located at the 5'-terminus of one strand. In one state, the strand is an antisense strand. In another state, the strand is a right strand. In a single-state sample, the internucleotide linkages of the thiophosphate or methylphosphonate are located at both the 5'-terminus and 3'-terminus of a strand. In a single-state sample, this strand is an antisense strand. In one state, the RNAi system contains 6 to 8 nucleotide linkages of thiophosphate. In one state, the antisense group comprises an internucleotide link of two thiophosphates located at the 5'-terminus and an internucleotide link of two thiophosphates located at the 3'-terminus, and the right-sense group comprises an internucleotide link of at least two thiophosphates located at the 5'-terminus or the 3'-terminus. In a single-state sample, the base pair located at the 5'-terminal 1 position of the antisense strand of the diploid is an AU base pair. In the first-state sample, the Y nucleotide contains a 2'-fluorine modification. In the first-state sample, the Y' nucleotide contains a 2'-O-methyl modification. In one sample, p'>0. In another sample, p'=2. In one state, q'=0, p=0, q=0, and the p' pendant nucleotides are complementary to the target mRNA. In another state, q'=0, p=0, q=0, and the p' pendant nucleotides are not complementary to the target mRNA. In one state, the positive nucleotide has a total of 21 nucleotides, and the antisense nucleotide has a total of 23 nucleotides. In a one-state sample, at least one n p It is linked to adjacent nucleotides via thiophosphate chains. In a one-state sample, all n p 'They are all linked to adjacent nucleotides via thiophosphate chains.' In one state, 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. In one state, the RNAi agent was AD-59743. In another state, the RNAi agent was AD-60940. On the one hand, the present invention provides a double-stranded RNAi agent that can inhibit the expression of TMPRSS6 in cells. This double-stranded RNAi agent comprises a positive and an antisense strand that form a double-stranded region. The positive nucleotide sequence comprises at least 15 adjacent nucleotides, wherein no more than 3 nucleotides are different from any of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; and the antisense nucleotide sequence comprises at least 15 adjacent nucleotides, wherein no more than 3 nucleotides are different from any of the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. Specifically, all nucleotides of the positive-sense term and all nucleotides of the anti-sense term contain modifications selected from the group consisting of 2'-O-methyl and 2'-fluorine modifications. Among them, the positive stock is composed of at least two nucleotide chains of thiophosphate at the 5'-terminus. Wherein, all the nucleotides of the antisense term and all the nucleotides of the antisense term contain modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluorine modifications. The antisense term comprises at least two nucleotide links of thiophosphate at the 5'-terminus and at least two nucleotide links of thiophosphate at the 3'-terminus. In this context, the positive stock is associated with one or more GalNAc derivatives that are linked to its 3'-terminus through a bivalent or trivalent linkage via a branch chain. In a single-state sample, all nucleotides of the positive-sense strand and all nucleotides of the anti-sense strand contain modifications. On the other hand, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (protein lyase-2) in cells, such as a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises complementary positive and negative strands, wherein the negative strand comprises a region complementary to a portion of the mRNA encoding TMPRSS6, wherein each strand is approximately 14 to approximately 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III): Justice: 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' Antonym: 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: i, j, k, and l are each independently 0 or 1; p, p', q and q' are each independently represented by 0 to 6; N a With N a Each 'represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; N b With N b Each 'represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified or unmodified or a combination thereof; n p n p '、n q and n q Each may or may not exist, and each represents a pendant nucleotide independently; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' are each independently representing a motif of three consecutive nucleotides modified in three identical ways, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modifier above is different from the modifier above Y, and N bThe modifier above 'Y' is different from the modifier above 'Y'; and The positive ligand binds to at least one ligand. In another aspect, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (protein lyase-2) in cells, such as a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises complementary positive and negative strands, wherein the negative strand comprises a region partially complementary to the mRNA encoding TMPRSS6, wherein each strand is approximately 14 to approximately 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III): Justice: 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' Antonym: 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: i, j, k, and l are each independently 0 or 1; n p n q and n q Each may or may not exist, and each represents a pendant nucleotide independently; p, q and q' are each independently represented by 0 to 6; n p >0, and at least one np' is linked to an adjacent nucleotide via a phosphate thioester link; N a With N a Each 'represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; N b With N b Each 'represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' are each independently representing a motif of three consecutive nucleotides modified in three identical ways, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modifier above is different from the modifier above Y, and N b The modifier above 'Y' is different from the modifier above 'Y'; and The positive ligand binds to at least one ligand. In another aspect, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (protein lyase-2) in cells, such as a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises complementary positive and negative strands, wherein the negative strand comprises a region partially complementary to the mRNA encoding TMPRSS6, wherein each strand is approximately 14 to approximately 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III): Justice: 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' Antonym: 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: i, j, k, and l are each independently 0 or 1; n p n q and n q Each may or may not exist, and each represents a pendant nucleotide independently; p, q and q' are each independently represented by 0 to 6; n p >0, and at least one np' is linked to an adjacent nucleotide via a phosphate thioester link; N a With N a Each 'represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; N b With N b Each 'represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' are each independently representing a motif of three consecutive nucleotides modified in three identical ways, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modifier above is different from the modifier above Y, and N b The modifier above 'Y' is different from the modifier above 'Y'; and The positive ligand is bound to at least one ligand, wherein the ligand is linked to one or more GalNAc derivatives via a divalent or trivalent linker of a branched chain. On the other hand, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (protein lyase-2) in cells, such as a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises complementary positive and negative strands, wherein the negative strand comprises a region complementary to a portion of the mRNA encoding TMPRSS6, wherein each strand is approximately 14 to approximately 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (III): Justice: 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' Antonym: 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: i, j, k, and l are each independently 0 or 1; n p n q and n q Each may or may not exist, and each represents a pendant nucleotide independently; p, q and q' are each independently represented by 0 to 6; n p >0, and at least one np' is linked to an adjacent nucleotide via a phosphate thioester link; N a With N a Each 'represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; N b With N b Each 'represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' are each independently representing a motif of three consecutive nucleotides modified in three identical ways, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; N b The modifier above is different from the modifier above Y, and N b The modifier above 'Y' is different from the modifier above 'Y'; The positive stock contains at least one thiophosphate ester chain; and The positive ligand is bound to at least one ligand, wherein the ligand is linked to one or more GalNAc derivatives via a divalent or trivalent linker of a branched chain. In another aspect, the present invention provides an RNAi agent capable of inhibiting the expression of TMPRSS6 (protein lyase-2) in cells, such as a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises complementary positive and negative strands, wherein the negative strand comprises a region complementary to a portion of the mRNA encoding TMPRSS6, wherein each strand is approximately 14 to approximately 30 nucleotides in length, and wherein the double-stranded RNAi agent is represented by formula (IIIa): Justice: 5' n p -N a -YY YN a -n q 3' Antonym: 3' n p '-N a '-Y'Y'Y'-N a '-n q '5' (IIIa) in: n p n q and n q Each may or may not exist, and each represents a pendant nucleotide independently; p, q and q' are each independently represented by 0 to 6; n p >0, and at least one np' is linked to an adjacent nucleotide via a phosphate thioester link; N a With N a Each 'represents an oligonucleotide sequence containing 0 to 25 nucleotides, which are modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent a motif of three consecutive nucleotides modified in three identical ways, wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; The positive stock contains at least one thiophosphate ester chain; and The positive ligand is bound to at least one ligand, wherein the ligand is linked to one or more GalNAc derivatives via a divalent or trivalent linker of a branched chain. 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, 10 and 12. On one hand, the present invention provides a composition comprising a modified antisense polynucleotide agent, wherein the agent inhibits the expression of TMPRSS6 in cells and comprises a sequence complementary to a sense sequence of a group consisting of sequences selected from any of Tables 1, 2, 4, 5, 8, 10 and 12, wherein the length of the polynucleotide is about 14 to about 30 nucleotides. The present invention also provides cells, vectors, host cells, and pharmaceutical compositions comprising double-stranded RNAi agents such as those of the present invention. In some cases, the RNAi agent is administered using a drug composition. In preferred formulations, the RNAi agent is administered in solution. In some of these formulations, the siRNA is administered in a non-buffered solution. In one formulation, the siRNA is administered in water. In other formulations, the siRNA is administered with a buffer solution such as acetate buffer, citrate buffer, prolamine buffer, carbonate buffer, or phosphate buffer, or a combination thereof. In some formulations, the buffer solution is phosphate-buffered saline (PBS). In one state, the drug composition further comprises a lipid formulation. In another state, the lipid formulation comprises LNP or XTC. In yet another state, the lipid formulation comprises MC3. On one hand, the present invention provides a method for inhibiting the expression of TMPRSS6 in cells. The method includes: exposing cells to 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 carrier of the present invention, or a pharmaceutical composition of the present invention; and maintaining the cells prepared in step (a) for a time sufficient to allow the mRNA transcript of the TMPRSS6 gene to be degraded, thereby inhibiting the expression of the TMPRSS6 gene in the cells. In one state, the cell line was in the subject's body. In a single state, the subject is a human. In one-state sample, TMPRSS6 expression was suppressed by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 100%. In another phenotype, the hepcidin gene expression line increased by at least approximately 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold. In another sample, serum hepcidin concentration increased by at least about 10%, about 25%, about 50%, about 100%, about 150%, about 200%, about 250%, or about 300%. In one state, serum iron concentration increased 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%. In another state, the percentage of transferrin saturation was 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%. On the other hand, the present invention provides a method for treating subjects suffering from TMPRSS6-mediated or related conditions. The method includes administering a therapeutically effective amount of the RNAi agent of the present invention, such as a double-stranded RNAi agent, a modified antisense polynucleotide agent of the present invention, a carrier of the present invention, or a pharmaceutical composition of the present invention, to the subject, thereby treating the subject. On one hand, the present invention provides a method for treating subjects suffering from TMPRSS6-related conditions. This method includes subcutaneously administering a therapeutically effective dose of a double-stranded RNAi agent to the subject. This double-stranded RNAi agent comprises a positive and an antisense strand that form a double-stranded region. The positive nucleotide sequence comprises at least 15 adjacent nucleotides, wherein no more than 3 nucleotides are different from any 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 nucleotide sequence comprises at least 15 adjacent nucleotides, wherein no more than 3 nucleotides are different from any 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. The antisense strand contains nucleotides that are essentially selected from the group consisting of 2'-O-methyl and 2'-fluorine modifications. The antisense term contains two nucleotide links of thiophosphate at the 5'-terminus and two nucleotide links of thiophosphate at the 3'-terminus. Specifically, all nucleotides of this positive stock contain modifications selected from the group consisting of 2'-O-methyl and 2'-fluorine modifications. Among them, the positive stock is composed of two nucleotide chains of thiophosphate at the 5'-terminus, and In this process, the positive stock is linked to one or more GalNAc derivatives that are connected to its 3'-terminus through a bivalent or trivalent chain linker via a branch chain, thereby treating the subject. In a single-state sample, all nucleotides of the positive-sense strand and all nucleotides of the anti-sense strand contain modifications. In a single state, the subject is a human. In one sample, the subject had a condition associated with iron overload, such as hereditary hemochromatosis, β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia), erythropoietinosis, Parkinson's disease, Alzheimer's disease, or Friedreich's ataxia. In one state, 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 a particular formulation, the double-stranded RNAi agent is administered at a dose of approximately 0.1 mg / kg, approximately 1.0 mg / kg, or approximately 3.0 mg / kg. In one state, the RNAi agent, such as a double-stranded RNAi agent, is administered subcutaneously or intravenously. In one state, the RNAi agent is administered in two or more doses. In a specific state, the RNAi agent is administered at intervals selected from the following groups: once every 12 hours, once every 24 hours, once every 48 hours, once every 72 hours, once every 96 hours, once every 7 days, and once every 14 days. In a particular state, the RNAi agent is administered once weekly for 2, 3, 4, 5 weeks, or longer. In another aspect, the present invention provides a method for treating iron overload-related conditions in a subject. This method includes administering a therapeutically effective amount of the RNAi agent of the present invention, such as a double-stranded RNAi agent or a carrier, to the subject, thereby treating the subject. In one state, the iron overload-related condition is hemochromatosis. In another state, the iron overload-related condition is thalassemia, such as β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia) or erythropoietinosis. In yet another state, the iron overload-related condition is a neurological disorder, such as Parkinson's disease, Alzheimer's disease, or Friedrich's ataxia. In one state, the subject is a primate or rodent. In another state, the subject is a human. In one state, 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. In one state, the RNAi agent, such as a double-stranded RNAi agent, is administered subcutaneously or intravenously. In one state, the RNAi agent is administered in two or more doses. In a specific state, the RNAi agent is administered at intervals selected from the following groups: once every 12 hours, once every 24 hours, once every 48 hours, once every 72 hours, once every 96 hours, once every 7 days, and once every 14 days. In one state, the administration resulted in a decrease in the subject's iron levels, ferritin levels, and / or transferrin saturation levels. In one state, the method further includes measuring the iron level in the subject's body. In one embodiment, the method of administering the iRNA agent (or pharmaceutical composition of the present invention) to a subject is performed in conjunction with the administration of additional drugs and / or other treatment methods. In another embodiment, the method of the present invention further comprises administering an iron chelating agent, such as deferiprone, deferoxamine, and deferasirox, to the subject. The present invention is further illustrated by the following detailed description and book. Figure 1 shows the relative levels of TMPRSS6 mRNA in the liver of wild-type mice after administration of a single dose of the iRNA agent AD-59743 at doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg. Figure 2 shows the relative levels of hepcidin mRNA in the liver of wild-type mice after administration of a single dose of the iRNA agent AD-59743 at doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg. Figures 3A through 3E show the liver TMPRSS6 mRNA levels (Figure 3A), hepcidin mRNA levels (Figure 3B), serum hepcidin levels (Figure 3C), total serum iron levels (Figure 3D), and transferrin saturation percentage levels (Figure 3E) at multiple time points in C57BL / 6 mice after a single subcutaneous injection of AD-60940 at doses of 0.3 mg / kg, 1.0 mg / kg, or 3.0 mg / kg, or after injection of PBS alone (control). Each data point represents the mean from three mice. The standard deviation of the mean is expressed in error bars. Figure 3F shows the relative liver TMPRSS6 mRNA concentration as a function of AD-60940 dose over 11 days post-administration. Each data point represents the maximum suppression of TMPRSS6 mRNA concentration observed at each dose level. The data conform to the Hill equation. Figure 4A illustrates a dosing regimen in mice, administering one dose weekly for three weeks, with mice sacrificed on day 21. Figure 4B shows the hepatic TMPRSS6 mRNA levels, hepcidin mRNA levels, and transferrin saturation percentage in C57BL / 6 mice after subcutaneous injection of 0.3 mg / kg, 1.0 mg / kg AD-60940, or PBS (control), according to the regimen shown in Figure 4A. Each bar represents the mean from three mice. The standard deviation of this mean is expressed as an error bar. Figure 4C shows the relative hepatic TMPRSS6 mRNA concentration as a function of the AD-60940 dose. This data conforms to the Hill equation. Figures 5A through 5D show the relationships between serum hepcidin concentration and relative TMPRSS6 mRNA levels (Figure 5A), transferrin saturation percentage and relative TMPRSS6 mRNA levels (Figure 5B), serum hepcidin concentration and relative hepcidin mRNA levels (Figure 5C), and transferrin saturation percentage and serum hepcidin concentration (Figure 5D). Figure 6 shows the relative levels of TMPRSS6 mRNA in the liver of C57BL / 6 mice after a single subcutaneous administration of the indicated iRNA agent at a dose of 3 mg / kg or PBS (control). The bars represent the mean from three mice, and the error bars represent the standard deviation of that mean. Figure 7 shows the relative levels of TMPRSS6 mRNA in the liver of C57BL / 6 mice after 3 weeks of weekly subcutaneous administration of the indicated iRNA agent at doses of 0.3 mg / kg or 1.0 mg / kg, or PBS (control). The bars represent the mean from three mice, and the error bars represent the standard deviation of that mean. Figure 8 shows the nucleotide sequence of Homo sapiens TMPRSS6 (SEQ ID NO: 1). Figure 9 shows the nucleotide sequence of TMPRSS6 in the house mouse (SEQ ID NO: 2). Figure 10 shows the nucleotide sequence of the TMPRSS6 rat (SEQ ID NO: 3). Figure 11 shows the nucleotide sequence of rhesus macaque TMPRSS6 (SEQ ID NO: 4). Figure 12 shows the nucleotide sequence (SEQ ID NO: 5) of the rhesus macaque TMPRSS6. Figure 13 shows the reverse complementary sequence of SEQ ID NO: 1 (SEQ ID NO: 6). Figure 14 shows the reverse complementary sequence of SEQ ID NO: 2 (SEQ ID NO: 7). Figure 15 shows the reverse complementary sequence of SEQ ID NO: 3 (SEQ ID NO: 8). Figure 16 shows the reverse complementary sequence of SEQ ID NO: 4 (SEQ ID NO: 9). Figure 17 shows the reverse complementary sequence of SEQ ID NO: 5 (SEQ ID NO: 10). This invention provides compositions comprising RNAi agents, such as double-stranded iRNA agents, that target TMPRSS6. This invention also provides methods for inhibiting TMPRSS6 expression and treating TMPRSS6-related conditions such as β-thalassemia or hemochromatosis using the compositions of this invention. TMPRSS6 plays a crucial role in iron homeostasis as an inhibitor of HAMP gene expression. The HAMP gene encodes hepcidin, a central regulator of iron homeostasis. Hepcidin binds to the iron export protein ferrotransferrin 1 (FPN1), primarily located in absorptive intestinal epithelial cells, hepatocytes, and macrophages. Hepcidin binding to the extracellular domain of FPN1 leads to its internalization and degradation, thereby reducing intestinal iron absorption and iron release from macrophages and hepatocytes. HAMP gene expression is stimulated in response to iron via a bone-forming protein (BMP) / transforming growth factor (SMAD)-dependent signaling cascade mediated by the BMP-coreceptor hepcidin regulatory protein (HJV). TMPRSS6's key role in HAMP regulation lies in its inhibition of BMP-mediated HAMP upregulation. TMPRSS6 inhibits BMP-mediated HAMP upregulation by cleaving the BMP co-receptor HJV, which is essentially responsible for BMP-mediated HAMP upregulation; thereby preventing BMP from signaling, SMAD from translocating to the nucleus, and HAMP from being transcribed and activated. Several studies in humans and mice have confirmed the role of TMPRSS6 in HAMP regulation and iron homeostasis (Du et al. Science 2008, Vol. 320, pp. 1088-1092; Folgeras et al. Blood 2008, Vol. 112, pp. 2539-45). Studies have shown that functional loss due to mutations in TMPRSS6 can lead to increased hepcidin expression, resulting in hereditary iron deficiency anemia, also known as iron-resistant iron deficiency anemia (IRIDA) (Finberg. Seminars in Hematology 2009, Vol. 46, pp. 378-86). IRIIDA is characterized by elevated hepcidin levels, microcytic hypochromic anemia, low mean corpuscular volume (MCV), low transferrin saturation, extremely poor oral iron absorption, and incomplete response to parenteral iron. However, loss of function in positive regulators of HAMP (such as BMP1, BMP4, and HFE) has been shown to downregulate hepcidin expression and cause iron overload syndromes (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 syndromes collectively known as hereditary hemochromatosis (HH), in anemias characterized by massive aplastic function, and in iron overload (secondary hemochromatosis) such as intermediate β-thalassemia (TI), despite elevated serum iron concentrations and iron reserves, hepcidin levels are low. A mouse model of intermediate β-thalassemia has shown that the loss of TMPRSS6 expression leads to increased hepcidin levels (Finberg 2010 Oral Presentation: "TMPRSS6, an inhibitor of Liver 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). This invention discloses iRNA agents, compositions, and methods for modulating the expression of the TMPRSS6 gene. In certain states, the use of TMPRSS6-specific iRNA agents reduces or inhibits the expression of TMPRSS6, resulting in increased HAMP expression and decreased serum iron levels. Therefore, the inhibition of TMPRSS6 gene expression or activity using the iRNA compositions proposed in this invention may be a useful approach for treatment aimed at reducing iron levels in subjects. This inhibition may be useful for treating iron overload-related conditions such as hemochromatosis or thalassemia, such as β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia). I. Definition To better understand this invention, certain terms are first defined. Furthermore, it should be noted that whenever a parameter value or range is referenced, it is assumed that the values ​​and ranges between the referenced values ​​are also part of this invention. The indefinite article “one” used in this article refers to one or more of its grammatical objects (i.e., at least one). For example, “element” means one element or more than one element, such as a plural of elements. The term "including" as used in this article refers to the phrase "including, but not limited to," and is used interchangeably with the latter. Unless otherwise specified in the context, the term "or" as used herein refers to the term "and / or" and is used interchangeably with the latter. In this article, "TMPRSS6" refers to the type II serous protease (TTSP) gene or protein. TMPRSS6 is also known as protein lyase-2, IRIDA (iron-resistant iron deficiency anemia), transmembrane protease serine 6, type II transmembrane serine protease 6, and membrane-bound intercalating serine protease protein lyase-2. TMPRSS6 is a type II transmembrane serine protease protein with a length of approximately 899 amino acids. TMPRSS6 contains multiple domains, such as a short endo domain, a transmembrane domain, a sea urchin sperm protein / enteropeptidase domain / agrin (SEA) domain, two complementary factor / sea urchin embryo growth factor / BMP domains (CUB), three class a LDL-R domains (LDLa), and a trypsin-like serine protease domain with a conserved His-Asp-Ser triplet (HDS). The term "TMPRSS6" includes human TMPRSS6, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. 56682967 (GI: 56682967); mouse TMPRSS6, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. 125656151; rat TMPRSS6, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. 194474097; and rhesus macaque TMPRSS6, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession Nos. XM_001085203.2 (GI: 297260989) and XM_001085319.1 (GI: 109094061). Other examples of AGT mRNA sequences can be easily obtained from publicly available databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website. The term "TMPRSS6" as used in this article also refers to naturally occurring DNA sequence variations in the TMPRSS6 gene, such as single nucleotide polymorphisms (SNPs) within the TMPRSS6 gene. Illustrative SNPs can be found in the open dbSNP database at www.ncbi.nlm.nih.gov / projects / SNP. The term "target sequence" as used in this article refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the TMPRSS6 gene, including mRNA as a primary transcription product of RNA processing. As used in this article, "a strand containing a sequence" refers to an oligonucleotide, which is a nucleotide chain containing a sequence revealed by using standard nucleotide nomenclature. "G", "C", "A", and "U" typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. In this document, "T" and "dT" are used interchangeably and refer to deoxyribonucleotides, where the nucleic acid base is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the terms "ribonucleotide," "nucleotide," or "deoxyribonucleotide" can also refer to modified nucleotides, as further detailed below, or to substitutions. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing characteristics of the oligonucleotide containing the nucleotide with the substitution. For example, but not limited to, nucleotides containing inosine as their base can be base-paired with nucleotides containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequences of the present invention, nucleotides containing uracil, guanine, or adenine can be replaced by nucleotides containing inosine, etc. Sequences containing such substitutions are typical of the present invention. In this article, the terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" are used interchangeably and refer to agents containing RNA as defined herein, which mediate the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process called RNA interference (RNAi). This iRNA regulates, for example, inhibits, the expression of TMPRSS6 in cells such as those of subjects, including mammalian subjects. In one embodiment, the RNAi agent of this invention is a single-stranded RNA that reacts with a target RNA sequence, such as the TMPRSS6 target mRNA sequence, to guide the cleavage of the target RNA. Not bound by theory, it is generally believed that long double-stranded RNA introduced into cells is broken into siRNA by a type III nuclease called clectinase (Sharp et al. (2001) Genes Dev. 15: 485). Clectinase, a ribonuclease-III-like enzyme, processes dsRNA into short interfering RNA of 19 to 23 base pairs characterized by two 3' hangers (Bernstein, et al., (2001) Nature 409: 363). Subsequently, these siRNAs are incorporated into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strands to guide target recognition (Nykanen, et al., (2001) Cell 107: 309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15: 188). Therefore, one aspect of the invention relates to single-stranded RNA (siRNA) produced in cells that promotes the formation of RISC complexes to influence the silencing of target genes such as the TMPRSS6 gene. Accordingly, the term "siRNA" is also used herein to refer to the aforementioned RNAi. In another embodiment, the RNAi agent may be a single-stranded siRNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which subsequently cleaves the target mRNA. These single-stranded siRNAs typically have 15 to 30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are disclosed in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of which are incorporated herein by reference. Any antisense nucleotide sequence disclosed herein may be used as a single-stranded siRNA, either as disclosed herein or as chemically modified by the methods disclosed in Lima et al., (2012) Cell 150:883-894. In another embodiment, the present invention provides a single-stranded antisense oligonucleotide molecule targeting 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 inhibit translation stoichiometrically by pairing with the bases of the mRNA and physically blocking the translation mechanism, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, the single-stranded antisense oligonucleotide molecule can inhibit the target mRNA by hybridizing to the target and cleaving the target via an RNaseH cleavage event. The single-stranded antisense oligonucleotide molecule can be about 10 to about 30 nucleotides in length and has a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule may comprise a sequence of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any of the antisense nucleotide sequences disclosed herein, such as those provided in any of Tables 1, 2, 4, 5, 8, 10 and 12, or in combination with any target site disclosed herein. Such single-stranded antisense oligonucleotide molecules may comprise modified RNA, DNA, or a combination thereof. In another embodiment, the "iRNA" used in the components, uses, and methods of this invention refers to double-stranded RNA, and herein refers to a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or simply "dsRNA." The term "dsRNA" refers to a nucleic acid molecule having a double helix containing two antiparallel and substantially complementary nucleic acid strands, indicating that it has "sense" and "antisense" orientation relative to the target RNA, i.e., the TMPRSS6 gene. In some embodiments of this invention, double-stranded RNA (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism, which herein refers to RNA interference or RNAi. Typically, most nucleotides in each strand of a dsRNA molecule are ribonucleotides, but as disclosed in detail herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, as used herein, "RNAi agent" may include chemically modified ribonucleotides; RNAi agents may include substantial modifications at multiple nucleotides. Such modifications may include all types disclosed herein or known in the art. For the purposes of this specification and the claims, any such modifications, as used in siRNA-type molecules, are covered by the term "RNAi agent". The two strands forming the double helix can be different parts of a larger RNA molecule, or they can be independent RNA molecules. If the two strands are parts of a larger molecule and are therefore linked by a non-interrupted nucleotide between the 3' end of one strand and the 5' end of the corresponding strand forming the double helix, the linked RNA strand is referred to as a "hairpin loop." If the two strands are covalently linked by means other than a non-interrupted nucleotide between the 3' end of one strand and the 5' end of the corresponding strand forming the double helix, the linking structure is referred to as a "linkage base." The RNA strands can 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 any dangling overhangs present in the double helix. In addition to the double helix structure, RNAi agents may contain one or more nucleotide overhangs. In one state, the RNAi agent of this invention is a dsRNA of 24 to 30 nucleotides, which reacts with a target RNA sequence, such as the TMPRSS6 target mRNA sequence, to guide the cleavage of the target RNA. To avoid being bound by theory, long double-stranded RNA introduced into the cell is broken into siRNA by a type III nuclease called clectinase (Sharp et al. (2001) Genes Dev. 15: 485). Clectinase, a ribonuclease-III-like enzyme, processes dsRNA into short interfering RNA of 19 to 23 base pairs characterized by two 3' overhangs (Bernstein, et al., (2001) Nature 409: 363). Subsequently, the siRNA is incorporated into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15:188). In this document, "nucleotide overhang" refers to an unpaired nucleotide or nucleotide protruding from the double helix structure of an RNAi agent when the 3' end of one strand extends beyond the 5' end of the other, or vice versa. "Blunt" or "blunt end" means that the ends of the double-stranded RNAi agent do not contain unpaired nucleotides, i.e., there is no nucleotide overhang. A "blunt-end" RNAi agent is a double-stranded dsRNA, meaning that there is no nucleotide overhang at either end of the molecule. The RNAi agents of this invention include RNAi agents with a nucleotide overhang at one end (i.e., an agent with one overhang and one blunt end) or with nucleotide overhangs at both ends. The term "antisense strand" refers to a strand of a double-stranded RNAi agent, which includes a region substantially complementary to the target sequence (e.g., human TMPRSS6 mRNA). In this document, the term "region partially complementary to the mRNA encoding thyroxine" refers to a region of the antisense strand substantially partially complementary to the TMPRSS6 mRNA sequence. If the complementary region is not perfectly complementary to the target sequence, the mismatch is most tolerable in the terminal regions, and if an incomplete match exists, it is typically located in one or more terminal regions, such as within 6, 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends. The term "positive stock" as used in this article refers to the stock of dsRNA, which includes regions that are substantially complementary to the region of the antisense stock. As used herein, the term "cleavage region" refers to the region immediately adjacent to a cleavage site. The cleavage site is the site where a cleavage occurs on the target. In some samples, the cleavage region comprises three bases located at one end of the cleavage site and immediately adjacent to it. In some specific samples, the cleavage region comprises two bases located at one end of the cleavage site and immediately adjacent to it. In some samples, the cleavage site specifically occurs at the binding site of the 10th and 11th nucleotides of the antisense strand, and the cleavage region comprises the 11th, 12th, and 13th nucleotides. In this document, unless otherwise specified, when the term "complementarity" is used to describe a first nucleotide sequence associated with a second nucleotide sequence, it 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 specific conditions to form a double helix structure, as understood by those skilled in the art. For example, such conditions can be stringent, including: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C, for 12 to 16 hours, followed by washing. Other conditions may be applied, such as physiologically relevant conditions that may be encountered in vivo. For example, complementary sequences are sufficient to enable relevant nucleic acid functions, such as RNAi. Those skilled in the art will be able to determine the most suitable composition of conditions for testing the complementarity of two sequences based on the final application of the hybridized nucleotides. When the nucleotides of the first nucleotide sequence and the nucleotides of the second nucleotide sequence have base pairings along the entire length of the first and second nucleotide sequences, the sequences are considered "perfectly complementary" to each other. However, in this document, if the first sequence is referred to as "substantially complementary" to the second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than four, three, or two mismatched base pairs after hybridization, while retaining their hybridization ability under the conditions most relevant to their final application. However, if two oligonucleotides are designed to form one or more single-stranded dangling sequences after hybridization, such dangling sequences should not be considered mismatched when determining complementarity. For example, a dsRNA containing one 21-nucleotide oligonucleotide and another 23-nucleotide oligonucleotide, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be considered "perfectly complementary" for the purposes disclosed herein. In this document, "complementary" sequences may also include, or be formed entirely from, the following: non-Watson-Crick base pairs within the range required for their hybridization and / or base pairs formed from non-natural and modified nucleotides. These non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing. In this article, the terms “complementary,” “fully complementary,” and “substantially complementary” can be used to describe the base pairing between the sense and antisense strands of dsRNA, or the pairing between the antisense strand of dsRNA and the target sequence, and can be understood from the context in which they are used. In this article, a polynucleotide that is "at least partially substantially complementary" to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an important mRNA (e.g., the mRNA encoding TMPRSS6), including a 5' UTR, an open reading frame (ORF), or a 3' UTR. For example, if a polynucleotide is substantially complementary to an uninterrupted portion of the mRNA encoding TMPRSS6, then the sequence is at least partially complementary to the TMPRSS6 mRNA. In this article, the term "inhibition" is used interchangeably with "reduction," "silence," "adjustment," "suppression," and other similar terms, and includes inhibition at any level. In this text, the phrase "suppressing the expression of TMPRSS6" includes the suppression of the expression of any TMPRSS6 gene (e.g., for example, the mouse TMPRSS6 gene, the rat TMPRSS6 gene, the monkey TMPRSS6 gene, or the human TMPRSS6 gene) and its variants (e.g., naturally occurring variants), or TMPRSS6 gene mutants. Therefore, the TMPRSS6 gene can be a wild-type TMPRSS6 gene, a mutant TMPRSS6 gene, or a transgenic TMPRSS6 gene in a normally manipulated cell, cell group, or organism context. "Suppression of TMPRSS6 gene expression" includes any level of suppression of TMPRSS6 gene expression, such as at least partial suppression of TMPRSS6 gene expression, such as suppression of 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%. TMPRSS6 gene expression can be assessed based on the levels of any variable associated with TMPRSS6 gene expression, such as TMPRSS6 mRNA levels, TMPRSS6 protein levels, hepcidin mRNA levels, hepcidin protein levels, or iron levels in tissues or serum. Inhibition can be assessed by comparing the absolute or relative levels of one or more of these variables to a decrease in control levels. This control level can be any type of control level used in this art, such as pre-dose baseline levels, or levels measured in similar subjects, cells, or samples that are untreated or controlled (e.g., for example, a control treated only with a buffer solution or a control treated with no active agent). In this document, the phrase "contacting cells with a double-stranded RNAi agent" includes contacting cells by any possible means. Contacting cells with a double-stranded RNAi agent includes contacting cells in vitro with the RNAi agent or contacting cells in vivo with the RNAi agent. This contact can be direct or indirect. Thus, for example, the method can be performed alone to physically contact the RNAi agent with the cells, or the RNAi agent can be placed in a situation that will allow or cause its subsequent contact with the cells. For example, in vitro cell contact can be achieved by culturing cells with an RNAi agent. In vivo cell contact can be achieved by injecting the RNAi agent into or near the tissue containing the cells; or by injecting the RNAi agent into another area, bloodstream, or subcutaneous space, thus allowing the agent to subsequently reach the tissue containing the cells to be contacted. For example, the RNAi agent may contain a ligand such as GalNAc. 3. A ligand and / or coupling with the latter directs the RNAi agent to a site of interest, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. In connection with these methods of the present invention, cells can be contacted with the RNAi agent in vitro and subsequently transplanted into subjects. In this article, "patient" or "subject" is intended to include humans or non-human animals, preferably mammals such as humans or monkeys. The subject or patient is preferably human. In this article, "TMPRSS6-related conditions" are intended to include any condition that can be treated or prevented by inhibiting the expression of TMPRSS6, or whose symptoms can be alleviated. In some cases, these TMPRSS6-related conditions are also associated with iron overload, the symptoms of which are typically elevated iron levels or iron dysregulation. For example, iron overload can be caused by, for instance, hereditary conditions; elevated dietary iron intake; or non-oral iron overload, including intravenous iron overload and transfusion iron overload. TMPRSS6-related conditions include, but are not limited to, hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis, sideroblastic anemia, hemolytic anemia, dyserythropoietic anemia, sickle cell anemia, heme disease, thalassemia (e.g., β-thalassemia and α-thalassemia), chronic liver disease, porphyria cutanea tarda, erythropoietinosis, iron-transfer protein anemia, hereditary tyrosinemia, brain-hepatorenal syndrome, idiopathic pulmonary hemosiderosis, and renal hemosiderosis. TMPRSS6-related conditions include those associated with oral iron overdose, transfusion iron overload, and intravenous iron overdose. TMPRSS6-related conditions also include conditions with symptoms associated with or caused by iron overload. These symptoms include an increased risk of liver disease (cirrhosis, liver cancer), heart attack or heart failure, diabetes, osteoarthritis, osteoporosis, metabolic syndrome, hypothyroidism, hypogonadism, and in some cases, premature death. In another category, TMPRSS6-related conditions include neurodegenerative diseases associated with iron overload and / or iron dysregulation, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Friedrich's ataxia, epilepsy, and multiple sclerosis. Administration of TMPRSS6-targeting iRNAs, such as those listed in any of Tables 1, 2, 4, 5, 8, 10, and 12, can treat one or more of these symptoms or prevent the development or progression of diseases or conditions exacerbated by increased iron levels. In the homozygous state, TMPRSS6-related disease is β-thalassemia. β-thalassemia is any of a group of inherited diseases characterized by a gene defect in the synthesis of the β-hemoglobin chain. In the homozygous state, β-thalassemia ("severe thalassemia") causes severe transfusion-dependent anemia. In the heterozygous state, the β-thalassemia phenotype ("mild thalassemia") causes mild to moderate microcytic anemia. Intermediate thalassemia is a type of β-thalassemia that causes a clinical severity of the disease that falls somewhere between the mild symptoms of mild β-thalassemia and severe β-thalassemia. Clinical diagnosis is made when the patient maintains a satisfactory hemoglobin level of at least 6 to 7 g / dL at the time of diagnosis without the need for routine blood transfusions. In one phenotype, β-thalassemia is classified as severe thalassemia. In the other phenotype, β-thalassemia is classified as intermediate thalassemia. In this article, "therapeutic effective dose" is intended to include an amount of RNAi agent sufficient to effectively treat (e.g., weaken, alleviate, or maintain the existing disease, or one or more symptoms of the disease) when administered to a patient for the treatment of TMPRSS6-related disease. This "therapeutic effective dose" may vary depending on the RNAi agent, how it is administered, the disease and its severity, and the patient's medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by TMPRSS6 manifestations, type of prior or combination therapy (if any), and other independent characteristics. In this article, "effective preventive dose" is intended to include an amount of RNAi agent sufficient to prevent or reduce the disease or one or more symptoms of the disease when administered to subjects who have not yet experienced or exhibited symptoms of TMPRSS6-related disease but may be susceptible to the disease. Reducing the disease includes slowing its progression or decreasing the severity of subsequent disease development. This "effective preventive dose" may vary depending on the RNAi agent, how it is administered, the level of disease risk, and the patient's medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by TMPRSS6 manifestations, type of prior or combination therapy (if any), and other independent characteristics. "Therapeutic effective dose" or "prophylactic effective dose" also includes an amount of RNAi agent that produces certain desired local or synthetic effects with any therapeutically acceptable benefit / risk ratio. The RNAi agent used in the method of the present invention can be administered in an amount sufficient to produce such a therapeutically acceptable benefit / risk ratio. In this document, the term "sample" includes aggregates of similar fluids, cells, or tissues isolated from a subject, or tissues present within the subject's body. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, eye discharge, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may originate from a specific organ, a portion of an organ, or fluids or cells within such organs. In some cases, the sample may originate from the liver (e.g., the whole liver, or certain sections of the liver, or certain types of cells within the liver, such as hepatocytes). In a preferred case, "sample from the subject" refers to blood or plasma drawn from the subject. In a further case, "sample from the subject" refers to liver tissue (or its sub-fractions) derived from the subject. II. iRNAs of the present invention This article discloses modified double-stranded RNAi agents that inhibit the expression of the TMPRSS6 gene in cells, such as in subjects, mammals, for example, in humans with TMPRSS6-related conditions such as β-thalassemia (e.g., severe β-thalassemia and intermediate β-thalassemia) or hemochromatosis; and the uses of such double-stranded RNAi agents. Accordingly, the present invention provides chemically modified double-stranded RNAi agents that can inhibit the expression of a target gene (i.e., the TMPRSS6 gene) in vivo. In some aspects of the present invention, substantially all nucleotides of the iRNA of the present invention are modified. In one embodiment of the present invention, all nucleotides of the iRNA of the present invention are modified. The iRNA of the present invention described as "substantially all nucleotides modified" is mostly, but not entirely, modified, and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides. This RNAi agent comprises a positive and an antisense strand. Each strand of the RNAi agent can be between 12 and 30 nucleotides in length. For example, each strand can be between 14 and 30 nucleotides in length, between 17 and 30 nucleotides in length, between 19 and 30 nucleotides in length, between 25 and 30 nucleotides in length, between 27 and 30 nucleotides in length, between 17 and 23 nucleotides in length, between 17 and 21 nucleotides in length, between 17 and 19 nucleotides in length, between 19 and 25 nucleotides in length, between 19 and 23 nucleotides in length, between 19 and 21 nucleotides in length, between 21 and 25 nucleotides in length, or between 21 and 23 nucleotides in length. The positive and negative strands typically form a double-stranded RNA (dsRNA), also referred to herein as an "RNAi agent." The double-stranded region of an RNAi agent can be 12 to 30 nucleotide pairs in length. For example, the double-stranded region can be between 14 and 30 nucleotide pairs, between 17 and 30 nucleotide pairs, between 27 and 30 nucleotide pairs, between 17 and 23 nucleotide pairs, between 17 and 21 nucleotide pairs, between 17 and 19 nucleotide pairs, between 19 and 25 nucleotide pairs, between 19 and 23 nucleotide pairs, between 19 and 21 nucleotide pairs, between 21 and 25 nucleotide pairs, or between 21 and 23 nucleotide pairs. In another example, the double helix region is selected from the lengths of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides. In one state, the RNAi agent may contain one or more overhanging regions and / or capping groups located at the 3'-end, 5'-end, or both ends of one or two strands. The overhangs may be 1 to 6 nucleotides in length, for example, 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. These overhangs may result in one strand being longer than the other, or two strands of the same length swinging together. The overhang may mismatch with the target mRNA, or it may be complementary to the target gene sequence, or it may be another sequence. The first and second strands may also form a hairpin loop by additional base bonding, or by other non-base binding sites. In a single-state sample, the nucleotides in the dangling region of the RNAi agent can each be independently modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be a dangling sequence located at either end of any strand. This dangling sequence may mismatch with the target mRNA, or it may be complementary to the target gene sequence, or it may be another sequence. The 5'- or 3'-hanging structures located on the positive, antisense, or both strands of the RNAi agent can be phosphorylated. In some states, the hanging structure contains two nucleotides with a phosphate thioester between them, wherein the two nucleotides may be the same or different. In one state, the hanging structure is located at the 3' end of the positive, antisense, or both strands. In one state, the 3'-hanging structure is located in the antisense strand. In one state, the 3'-hanging structure is located in the positive strand. The RNAi agent may contain only a single dangling endplate, which enhances the interfering activity of the RNAi without affecting its overall stability. For example, the single-stranded dangling endplate may be located at the 3' end of the positive strand, or at the 3' end of the negative strand. The RNAi may also have a blunt end at the 5' end of the negative strand (or the 3' end of the positive strand), or vice versa. Typically, the negative strand of an RNAi has a nucleotide dangling endplate at the 3' end, while its 5' end is blunt. Although not wishing to be bound by theory, this asymmetric blunt endplate at the 5' end and dangling endplate at the 3' end of the negative strand helps guide the strand loading into the RISC process. Any nucleic acid that is a feature of this invention can be synthesized and / or modified by methods known in the art, such as those disclosed in "Current protocols in nucleic acid chemistry," Beaucage, S. Letal. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications such as 5'-end modifications (phosphorylation, conjugation, anti-linking) or 3'-end modifications (conjugation, DNA nucleotides, anti-linking, etc.); base modifications such as substitution with a stabilized base, an unstable base, or a base paired with an expanded repertoire, base removal (debaseted nucleotides), or conjugated bases; sugar modifications (e.g., modifications at the 2' or 4' position) or sugar substitutions; and / or backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of iRNA compounds that can be used in the isomorphic samples disclosed herein include, but are not limited to, RNAs containing a modified backbone or non-natural internucleotide chains. RNAs with a modified backbone include, in particular, those whose backbone does not contain phosphorus atoms. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs lacking phosphorus atoms in their internucleotide backbone may also be considered oligonucleotides. In some isomorphic samples, the modified iRNA will have phosphorus atoms in its internucleotide backbone. Modified RNA backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methylphosphonates, and other phosphonic acid alkyl esters including 3'-alkylphosphonates and chiral phosphonates, phosphonites, aminophosphates including 3'-aminophosphonamidates and aminoalkylphosphonamidates, thionophosphoramides, thioalkylphosphonates, thiocarbonyl phosphate triesters, and boroalkyl phosphates, their 2'-5' linkage analogs, and those with opposite polarity, wherein adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. They also include various salts, mixed salts, and free acid forms. Representative U.S. patents illustrating the preparation of phosphorus-containing chains include, but are not limited to, Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, and 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. The entire contents of Nos. 6,444,423, 6,531,590, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and US Pat RE39464 are incorporated herein by reference. The modified RNA backbone, excluding phosphorus atoms, has a backbone formed by: short-chain alkyl or cycloalkyl links between nucleosides; mixed links of heteroatoms and alkyl or cycloalkyl groups between nucleosides; or one or more short-chain heteroatoms or heterocyclic links between nucleosides. This includes those having: N-morpholine links (partially formed from the glycosidic moieties of nucleosides); siloxane backbones; thioether, urethane, and urethane backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; backbones containing alkyl groups; aminosulfonate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH groups. Two groups of people. Representative U.S. patents teaching the preparation of the aforementioned oligonucleotides include, but are not limited to, Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, and 5, The entire contents of Nos. 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 are incorporated herein by reference. In other forms, suitable RNA mimicry systems are intended for use in iRNAs, wherein both the sugar and nucleoside internucleotide chains (i.e., the backbone) of these nucleotides are replaced with novel groups. These base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimicry exhibiting excellent hybridization properties, is a peptide nucleic acid (PNA). In PNA compounds, the glycosyl backbone of the RNA is replaced with an acetylamine-containing backbone, particularly aminoethylglycine. These nucleic acid bases are retained and directly or indirectly bonded to the nitrogen atom of the acetylamine portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of which are incorporated herein by reference. Other PNA compounds suitable for use in the iRNA of this invention are disclosed in publications such as Nielsen et al., Science, 1991, 254, 1497-1500. Certain state-forms characteristic of this invention include RNAs having a phosphate thioester backbone and a heteroatom backbone, the backbone being particularly referred to in U.S. Patent No. 5,489,677 cited above—CH. 2--NH--CH 2-、--CH 2--N(CH 3)--O--CH 2--[referred to as 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 represented as --O--P--O--CH 2--] and the backbone of U.S. Patent No. 5,602,240 cited above. In some morphologies, the RNA line characteristic herein has the N-morpholino backbone structure of U.S. Patent No. 5,034,506 cited above. Modified RNA may also contain one or more substituted sugar molecules. Such iRNAs, such as the dsRNAs characteristic of this document, 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, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C- groups. 1 to C 10 Alkyl or C 2 to C 10 Alkenyl and alkynyl groups. Examples of suitable modification systems include O[(CH2]2]2. 2) n O] m CH 3. O(CH) 2) n OCH 3. O(CH) 2)nNH 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, where n and m are 1 to approximately 10. In other states, dsRNA includes one of the following at the 2' position: C 1 to C 10 Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl alkyl groups, O-alkylaryl or O-aryl alkyl groups, SH, SCH 3. OCN, Cl, Br, CN, CF 3. OCF 3. SOCH 3. SO 2CH 3. ONO 2. NO 2. N 3. NH 2. Heterocyclic alkyl, heterocyclic aryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalating agent, group used to improve the pharmacokinetics of iRNA, or group used to improve the pharmacodynamics of iRNA, and other substituents with similar properties. In some states, the modification system includes 2'-methoxyethoxy(2'-O--CH) 2CH 2OCH 3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., alkoxy-alkoxy. Another example of an indicative modification is 2'-dimethylaminooxyethoxy, i.e., O(CH 2) 2ON(CH 3) 2-dimethylaminoethoxyethoxy (also known in this art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH 2--O--CH 2--N(CH 2) 2. Other modification systems include 2'-methoxy(2'-OCH) 3) 2'-Aminopropoxy (2'-OCH 2CH 2CH 2NH 2) and 2'-fluorine (2'-F). Similar modifications can also be made at other positions on the RNA of the iRNA, particularly at the 3' end nucleotide or at the 3' position of the dsRNA with the 2'-5' strand, and at the 5' position of the 5' end nucleotide. The iRNA can also have a glycomimetic such as a cyclobutyl derivative to replace the sugar in the furanopentoside. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, 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, and 5,5 Patents Nos. 67,811, 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 are sometimes shared with this application. The entire contents of each of the foregoing are incorporated herein by reference. iRNA may also include nucleobase modifications or substitutions (often abbreviated as "bases" in this art). In this article, "unmodified" or "natural" nucleobase systems include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobase systems include other synthetic and natural nucleobases, such as deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and 6-other alkyl derivatives of adenine and guanine, 2-propyl and 2-other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azycytosine, 6-azthymidine, 5-uracil (pseudouracil), 4-thiouracil, 8-halouracil, etc. Adenine, 8-haloguanine, 8-aminoadenine, 8-aminoguanine, 8-mercaptoadenine, 8-mercaptoguanine, 8-thioalkyladenine, 8-thioalkylguanine, 8-hydroxyadenine, 8-hydroxyguanine, other 8-substituted adenines and guanines, 5-halouracil, especially 5-bromouracil, 5-halocytosine, especially 5-bromocytosine, 5-trifluoromethyluracil, 5-trifluoromethylcytosine, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazoguanine, 7-deazoadenine, 3-deazoguanine, and 3-deazoadenine. Other nucleobase systems include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Certain of these nucleobase systems are particularly useful for increasing the binding affinity of the oligomeric compounds that are characteristic of this invention. These systems include 5-substituted pyrimidines; 6-azapyrimidines; and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the stability of the nucleic acid double helix by 0.6 to 1.2 °C (Sanghvi, YS, Crooke, STand Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is an exemplary base substitution; its effect is even more pronounced when combined with 2'-O-methoxyethyl sugar modification. Representative U.S. patents teaching the preparation of certain of the aforementioned modified nucleosides and other modified nucleosides include, but are not limited to, those 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, and 5,594,121. U.S. Patents Nos. 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 which are incorporated herein by reference. The RNA of iRNA can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribosomes, wherein the ribosome system contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose into a 3'-in-type configuration. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193). Representative U.S. patents teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patents No. 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 which are incorporated herein by reference. Effective stabilization modifications for RNA molecules include N-(acetylamydohexyl)-4-hydroxyproline (Hyp-C6-NHAc), N-hexyl-4-hydroxyproline (Hyp-C6), N-acetylamydohexylproline (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexyl)-4-hydroxyproline (Hyp-C6-amino), 2-eicosyl-uridine-3"-phosphate, and the reverse base dT (idT). This modification is disclosed in World Patent No. 2011 / 005861. A. Modified iRNA containing the motif of this invention In some embodiments of the present invention, the double-stranded RNAi agent of the present invention includes a chemically modified agent, such as that disclosed in U.S. Provisional Application No. 61 / 561,710, filed November 18, 2011, or in PCT / US2012 / 065691, filed November 16, 2012, the entire contents of which are incorporated herein by reference. As illustrated herein and Provisional Application No. 61 / 561,710, outstanding results can be obtained by introducing one or more motifs with three identical modifications on three consecutive nucleotides into the positive and / or antisense strands of an RNAi agent, particularly at or near the cleavage site. In some cases, the positive and antisense strands of the RNAi agent may be fully modified. If a modification pattern exists in the positive and / or antisense strands, the introduction of such modifications disrupts that pattern. The RNAi agent can bind to a GalNAc derivative ligand as desired, for example, to the positive strand. The resulting RNAi agent exhibits outstanding gene-silencing activity. More specifically, it has been surprisingly found that when the sense and antisense strands of a double-stranded RNAi agent are modified to have one or more motifs with 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 enhanced. In one state, the RNAi agent is a bibliophilic RNAi agent with a length of 19 nucleotides, wherein the sense strand contains at least one motif with three consecutive nucleotides at positions 7, 8, and 9 of the 5' end modified by three 2'-F molecules. The antisense strand contains at least one motif with three consecutive nucleotides at positions 11, 12, and 13 of the 5' end modified by three 2'-O-methyl molecules. In another embodiment, the RNAi agent is a bibliophilic RNAi agent with a length of 20 nucleotides, wherein the sense strand contains at least one motif with three consecutive nucleotides at positions 8, 9, and 10 of the 5' end modified by three 2'-F molecules. The antisense strand contains at least one motif with three consecutive nucleotides at positions 11, 12, and 13 of the 5' end modified by three 2'-O-methyl molecules. In another state, the RNAi agent is a bibliophilic RNAi agent with a length of 21 nucleotides, wherein the sense strand contains at least one motif with three consecutive nucleotides at positions 9, 10, and 11 at the 5' end modified by three 2'-F molecules. The antisense strand contains at least one motif with three consecutive nucleotides at positions 11, 12, and 13 at the 5' end modified by three 2'-O-methyl molecules. In one state, the RNAi agent comprises a 21-nucleotide positive strand and a 23-nucleotide antisense strand. The positive strand contains at least one motif of three consecutive nucleotides at positions 9, 10, and 11 of the 5' end modified with three 2'-F groups. The antisense strand contains at least one motif of three consecutive nucleotides at positions 11, 12, and 13 of the 5' end modified with three 2'-O-methyl groups. One end of the RNAi agent is blunt, and the other end contains a 2-nucleotide dangling ligature. Preferably, the 2-nucleotide dangling ligature is located at the 3' end of the antisense strand. When the 2-nucleotide dangling ligature is located at the 3' end of the antisense strand, two phosphate thioester nucleotide base chains may exist between the three terminal nucleotides. Two of these three nucleotides are the dangling nucleotides, and the third nucleotide pairs with the nucleotide following the dangling nucleotide. In one state, two additional phosphate thioester nucleotide chains are present between the terminal three nucleotides at the 5'-end of the positive and antisense strands of the RNAi agent. In one state, each nucleotide in the positive and antisense strands of the RNAi agent, including nucleotides that are part of the motif, is a modified nucleotide. In one state, each residue, for example, is independently modified with 2'-O-methyl or 3'-fluorine in an alternating motif. If desired, the RNAi agent may also contain a ligand (preferably GalNAc). 3). In one embodiment, the RNAi agent comprises a sense strand and an antisense strand. The RNAi agent comprises a first strand of at least 25 and at most 29 nucleotides in length, and a second strand of at most 30 nucleotides in length. The second strand has a motif where at least three consecutive nucleotides at positions 11, 12, and 13 at the 5' end are modified with three 2'-O-methyl groups. The 3' end of the first strand and the 5' end of the second strand form blunt ends, and the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end. The length of the double helix region is at least 25 nucleotides. When the RNAi agent is introduced into mammalian cells, the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of its length to reduce the expression of the target gene. Furthermore, the cleavage enzyme cleavage of the RNAi agent preferentially occurs on the siRNA containing the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, the RNAi agent may also include a ligand. In one state, the positive strand of the RNAi agent contains at least one motif that has been modified in three consecutive nucleotides with the same three modifications, and one of the motifs is located at the cleavage site of the positive strand. In one state, the antisense strand of the RNAi agent contains at least one motif with three identical modifications on three consecutive nucleotides, and one of the motifs is located at the cleavage site of the antisense strand. For RNAi agents with a double-helix region of 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically located near positions 10, 11, and 12 at the 5' end. Therefore, these three identically modified motifs can appear at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15, on the antisense strand, counting from the first nucleotide at the 5' end, or from the first paired nucleotide within the double-helix region at the 5' end. The cleavage site in the antisense strand can also vary depending on the length of the double-helix region of the RNAi from the 5' end. The positive strand of the RNAi agent may contain at least one motif located at the cleavage site of the strand, consisting of three identical modifications on three consecutive nucleotides; and the antisense strand may contain at least one motif located at or near the cleavage site of the strand, consisting of three identical modifications on three consecutive nucleotides. When the positive and antisense strands form a dsRNA double helix, the positive and antisense strands can be aligned such that one of the three nucleotide motifs on the positive strand overlaps with one of the three nucleotide motifs on the antisense strand by at least one nucleotide, that is, at least one of the three nucleotides of the motif in the positive strand forms a base pair with at least one of the three nucleotides in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap. In a single-state sample, the positive strand of the RNAi agent may contain more than one motif with three identical modifications on three consecutive nucleotides. The first motif may appear at or near the cleavage site of the strand, while the other motifs may be flanking modifications. Hereinafter, the term "flanking modification" refers to a motif that appears in a portion of the strand separated from or near the cleavage site of the same strand. The flanking modification may be adjacent to the first motif or separated from it by at least one or more nucleotides. When the motifs are adjacent to each other, their chemistry is distinct; when the motifs are separated by one or more nucleotides, their chemistry may be the same or different. Two or more flanking modifications may be present. For example, when two flanking modifications are present, each flanking modification may appear at the opposite end of the first motif at or near the cleavage site, or on either side of the lead motif. Similar to the positive strand, the antisense strand of this RNAi agent may contain more than one motif consisting of three identical modifications on three consecutive nucleotides, and at least one of these motifs is present at or near the cleavage site of the strand. This antisense strand may also contain one or more flanking modifications arranged similarly to those present on the positive strand. In a single-state sample, the flanking modifications on the sense or antisense strand of the RNAi agent typically do not include the first one or two terminal nucleotides at the 3'-end, 5'-end, or both ends of the strand. In another state, the flanking modifications on the sense or antisense strand of the RNAi agent typically do not include the first one or two paired nucleotides in the double helix region at the 3'-end, 5'-end, or both ends of the strand. When the sense and antisense strands of the RNAi agent each contain at least one flanking modification, the flanking modification can fall on the same end of the double helix region and have one, two or three nucleotide overlaps. When the positive and antisense strands of the RNAi agent each contain at least two flanking modifications, the positive and antisense strands can be aligned in the following manner: the two modifications from each strand fall into one end of the double helix region, with an overlap of one, two, or three nucleotides; the two modifications from each strand fall into the other end of the double helix region, with an overlap of one, two, or three nucleotides; the two modifications from each strand fall into their respective sides of the leading motif, and have an overlap of one, two, or three nucleotides within the double helix region. In a single-state sample, each nucleotide of the positive and antisense strands of the RNAi agent, including nucleotides that form part of the motif, may be modified. Each nucleotide may be modified in the same or different ways, including changes to one or more non-linked phosphodiester oxygens and / or one or more linked phosphodiester oxygens; changes to ribose components such as the 2'-hydroxyl group on the ribose; replacement of the entire phosphodiester body with a "dephosphorylated" linker; modification or replacement of native bases; and replacement or modification of the ribose-phosphodiester backbone. Because nucleic acids are polymers of subunits, most modifications occur at repetitive positions within the nucleic acid, such as modifications to bases, phosphodiester bodies, or non-linking Os of phosphodiester bodies. In some cases, the modification will appear at all target positions of the nucleic acid, but in most cases this is not the case. For example, a modification may appear only at the 3' or 5' end, or only in the terminal region, such as at the terminal nucleotide position of a strand or in the last 2, 3, 4, 5, or 10 nucleotides. A modification may appear in double-stranded regions, single-stranded regions, or both. A modification may appear only in double-stranded regions of RNA or only in single-stranded regions of RNA. For example, a phosphate thioester modification at a non-linking O position may appear only at one or both ends, or only in the terminal region, such as at the terminal nucleotide position of a strand or in the last 2, 3, 4, 5, or 10 nucleotides, or may appear in both double-stranded and single-stranded regions, especially at the ends. One or more 5' ends may be phosphorylated. To enhance stability, it is possible to include specific bases in the pendant, or to include modified nucleotides or nucleotide substitutes in a single-stranded pendant such as a 5' pendant or a 3' pendant, or both. For example, it is conceivable to include purine nucleotides in the pendant. In some states, all or some bases in the 3' or 5' pendant may be modified as disclosed herein. Modifications may include, for example, modifications to the 2' position of the ribose using techniques prior to this art, such as replacing the ribose of the nucleobase with a 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modified deoxyribonucleotide, and modifications to the phosphate group, such as thiophosphate modifications. The pendant need not be homologous to the target sequence. In one state, each residue of the positive and negative strands is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy, or 2'-fluorine. These strands may contain more than one modification. In one state, each residue of the positive and negative strands is independently modified with 2'-O-methyl or 2'-fluorine. At least two different modifications are typically present on the positive and negative stock. These two modifications may be 2'-O-methyl or 2'-fluoro, or others. In a single-state sample, the N a and / or N b The term "alternating motif" as used herein refers to a motif having one or more modifications, each modification appearing on a set of alternating nucleotides. These alternating nucleotides may be grouped by every other nucleotide, by every three nucleotides, or similar patterns. For example, if A, B, and C each represent a type of modification to the nucleotides, then the alternating motif could be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…", or "ABCABCABCABC…", etc. The types of modifications contained in the alternation motif can be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, then the alternation pattern, that is, the modifications on every other nucleotide, can be the same, but each of the positive or negative shares can be selected from several possibilities of modifications covered by the alternation motif, such as "ABABAB…", "ACACAC…", "BDBDBD…", or "CDCDCD…", etc. In one embodiment, the RNAi agent of the present invention comprises a modification pattern of alternating motifs on the positive strand that is shifted relative to the modification pattern of alternating motifs on the antisense strand. This shift can be as follows: a modified base of the nucleotide in the positive strand corresponds to a different modified base of the nucleotide in the antisense strand, and vice versa. For example, when the positive and antisense strands pair in a dsRNA double helix, within the double helix region, the alternating motif in the positive strand can extend from the 5' end of the strand towards the 3' end starting with "ABABAB", while the alternating motif in the antisense strand can extend from the 5' end of the strand towards the 3' end starting with "BABABA". As another example, within the double helix region, the alternating module in the positive stock can extend from the 5' end of the stock to the 3' end starting with "AABBAABB", while the alternating module in the negative stock can extend from the 5' end of the stock to the 3' end starting with "BBAABBAA". Therefore, there is a modification pattern of complete or partial displacement between the positive and negative stocks. In one state, the RNAi agent comprises a pattern in which the initial alternating motif of 2'-O-methyl and 2'-F modifications on the positive strand is shifted relative to the initial alternating motif of 2'-O-methyl and 2'-F modifications on the antisense strand. That is, the nucleotide modified with 2'-O-methyl on the positive strand pairs with the nucleotide modified with 2'-F on the antisense strand, and vice versa. Position 1 of the positive strand can be initiated by 2'-F modification, and position 1 of the antisense strand can be initiated by 2'-O-methyl modification. Introducing one or more motifs with three identical modifications on three consecutive nucleotides into the positive and / or negative strand interrupts the initial modification present in the positive and / or negative strand. This interruption of the modification pattern of the positive and / or negative strand by introducing motifs with three identical modifications on three consecutive nucleotides surprisingly enhances its gene silencing activity against the target gene. In a single-state sample, when a motif consisting of three identical modifications on three consecutive nucleotides is introduced into an arbitrary strand, the modifications to the nucleotides adjacent to the motif are different from the modifications to the motif itself. For example, the sequence portion containing the motif is "...N a YYYN b …”, where “Y” represents a modification of three consecutive nucleotides by three identical motifs, and “N” represents… a " and "N b The "" indicates a modification of the nucleotide adjacent to the "YYY" motif that differs from the "Y", where N a With N bThese can be the same or different modifiers. Alternatively, when flanking modifiers exist, N a and / or N b It may or may not exist. The RNAi agent may contain at least one internucleotide link of a phosphate thioester or methylphosphonate. This internucleotide link modification of the phosphate thioester or methylphosphonate may occur on any nucleotide at any position in the positive or negative strand, or both strands. For example, the internucleotide link modification may occur on each nucleotide of the positive and / or negative strand; the internucleotide link modification may each occur in an alternation pattern of the positive and / or negative strands; or the positive or negative strand may contain two internucleotide link modifications in an alternation pattern. The alternation pattern of the internucleotide link modification on the positive strand may be the same as or different from that on the negative strand, and the alternation pattern of the internucleotide link modification on the positive strand may have a shift relative to the alternation pattern of the internucleotide link modification on the negative strand. In one state, the RNAi system contains an internucleotide linkage modification of phosphate thioester or methylphosphonate in the dangling region. For example, the dangling region may contain two nucleotides with an internucleotide linkage of phosphate thioester or methylphosphonate between them. The internucleotide linkage modification may also involve linking the dangling nucleotide to the terminal paired nucleotides within the double helix region. For example, at least two, three, four, or all of the dangling nucleotides may be linked via an internucleotide linkage of phosphate thioester or methylphosphonate, and, if necessary, additional internucleotide linkages of phosphate thioester or methylphosphonate may be present to link the dangling nucleotide to the adjacent paired nucleotide. For example, if two of the three terminal nucleotides are dangling nucleotides and the third is a paired nucleotide adjacent to the dangling nucleotide, then at least two internucleotide linkages of phosphate thioester may exist between these three nucleotides. These terminal three nucleotides may be located at the 3' end of the antisense term, the 3' end of the righteous term, the 5' end of the antisense term, and / or the 5' end of the righteous term. In one state, the dangling nucleotides of the two nucleotides are located at the 3' end of the antisense strand, and there are two phosphate thioester internucleotide links between the three terminal nucleotides, wherein two of the three nucleotides are dangling nucleotides, and the third is a paired nucleotide adjacent to the dangling nucleotide. If necessary, the RNAi agent may additionally have two phosphate thioester internucleotide links between the three terminal nucleotides at both the 5' end of the positive and antisense strands. In a single-state sample, the RNAi system contains mismatches with the target, mismatches within the double helix, or combinations thereof. These mismatches can occur in overhanging regions or double-helix regions. Base pairs can be ranked based on their potential to promote dissociation or dissolution (e.g., the free energy of association or dissociation of a particular pair, the simplest way to examine such pairings on an independent pairing basis, but next neighbor analysis or similar analyses can also be used). From the perspective of promoting dissociation, the A:U system is superior to the G:C system; the G:U system is superior to the G:C system; and the I:C system is superior to the G:C system (I = inosine). Mismatches that are non-canonical pairings or non-canonical pairings (as disclosed elsewhere in this document) are superior to canonical pairings (A:T, A:U, G:C); and pairings that include universal bases are superior to canonical pairings. In one state, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the first part of the double helix region from the 5' end of the antisense strand, which is independently selected from the group consisting of: A:U, G:U, I:C, and mismatched base pairs, such as non-regular pairings or non-regular pairings or pairings including universal bases, to promote the dissociation of the double helix at the 5' end of the antisense strand. In one state, the nucleotide at the first position of the 5' end of the antisense strand within the double helix region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first, second, or third base pairs at the very beginning of the 5' end of the antisense strand within the double helix region is an AU base pair. For example, the first base pair at the 5' end of the antisense strand within the double helix region is an AU base pair. In a single-state sample, the sequence of positive stocks can be represented by equation (I): 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' (I) in: i and j are each independently 0 or 1; The p and q series are each independently represented by values ​​from 0 to 6; Every N a Each oligonucleotide sequence independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, and each sequence contains at least two different modified nucleotides; Every N b Each represents an oligonucleotide sequence containing 0 to 10 modified nucleotides independently; Every n p and n q The term "suspended nucleotide" is used to represent a single, independent nucleotide. Where, N b It does not have the same modifications as the Y series; and XXX, YYY, and ZZZ each independently represent a motif consisting of three identical modifications on three consecutive nucleotides. YYY is preferably a nucleotide that is entirely modified with 2'-F. In a single-state sample, N a and / or N b The system includes modifications for alternating patterns. In a single-state sample, the YYY motif appears at or near the cleavage site of the positive strand. For example, when the RNAi agent has a double helix region of 17 to 23 nucleotides in length, the YYY motif may appear at or near the cleavage site of the positive strand (e.g., at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12 or 11, 12, 13), counted starting from the first nucleotide at the 5' end; or, as needed, counted starting from the first paired nucleotide at the 5' end of the double helix region. In a single-state sample, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. This positive stock can therefore be expressed by the following formula: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib); 5' n p -N a-XXX-N b -YYY-N a -n q 3' (Ic); or 5' n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id). When the right stock is expressed by formula (Ib), N b This indicates 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 It can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. When the justice stock is represented as (Ic), N b This indicates 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 It can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. When the stock is represented as (Id), N b This indicates 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 It can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each X, Y, and Z can be the same as or different from each other. In other states, i is 0, j is 0, and the positive stock can be expressed by the following formula: 5' n p -N a -YYY-N a -n q 3' (Ia). When the right stock is represented by formula (Ia), each N a It can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. In a one-state sample, the antisense strand sequence of the RNAi can be represented by equation (II): 5' n q '-N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N a '-n p '3' (II) in: k and l are each independently 0 or 1; p' and q' are each independently represented by numbers from 0 to 6; Every N a 'It independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; Every N b 'It independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; Every n p 'and n q 'A single nucleotide is used to represent a pendant nucleotide;' Where, N b 'Y' does not have the same modifiers; as well as The X'X'X', Y'Y'Y', and Z'Z'Z' motifs each independently represent a motif consisting of three identical modifications on three consecutive nucleotides. In a single-state sample, N a 'and / or N b The system includes modifications for alternating patterns. The Y'Y'Y' motif appears at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a double helix region of 17 to 23 nucleotides in length, the Y'Y'Y' motif may appear at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, and be counted starting from the first nucleotide at the 5' end; or, if necessary, be counted starting from the first paired nucleotide at the 5' end of the double helix region. The Y'Y'Y' motif preferably appears at positions 11, 12, and 13. In the first-state sample, all nucleotides of the Y'Y'Y' pattern system are modified with 2'-OMe. In a single-state sample, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1. This antisig can therefore be expressed 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). When the antonymous stock is represented by equation (IIb), 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 'Can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.' When the antonymous stock system is expressed as equation (IIc), 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 'Can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.' When the antonymous stock is expressed as equation (IId), 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 'Can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. N b The preferred numbers are 0, 1, 2, 3, 4, 5, or 6. In other states, k is 0, l is 0, and the antisense can be expressed by the following formula: 5' n p' -N a' -Y'Y'Y'-N a' -n q' 3' (Ia). When the antisense series is expressed as equation (IIa), each N a 'It independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each X', Y', and Z' can be the same as or different from each other. The nucleotides of the positive and negative strands can each be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxy, or 2'-fluorine. For example, the nucleotides of the positive and negative strands can each be independently modified with 2'-O-methyl or 2'-fluorine. In particular, each X, Y, Z, X', Y', and Z' can represent 2'-O-methyl modification or 2'-fluorine modification. In a single-state sample, when the double helix region is 21 nt, the positive strand of the RNAi agent may contain YYY motifs appearing at positions 9, 10, and 11 of the strand, counted starting from the first nucleotide at the 5' end, or, as needed, counted starting from the first paired nucleotide at the 5' end of the double helix region; and the Y series indicates 2'-F modification. The positive strand may also contain XXX or ZZZ motifs at the opposite ends of the double helix region as flanking modifications; and XXX and ZZZ series each independently represent 2'-OMe modification or 2'-F modification. In one state, the antisense strand may contain Y'Y'Y' motifs appearing at positions 11, 12, and 13 of the strand, counted starting from the first nucleotide at the 5'-end, or, as needed, counted starting from the first paired nucleotide at the 5'-end of the double helix region; and Y' represents 2'-O-methyl modification. The antisense strand may also contain X'X'X' or Z'Z'Z' motifs at opposite ends of the double helix region as flanking modifications; and X'X'X' and Z'Z'Z' each independently represent 2'-OMe modification or 2'-F modification. The positive stock represented by any one of the above equations (Ia), (Ib), (Ic) and (Id) forms a double helix with the antisig represented by equations (IIa), (IIb), (IIc) and (IId). Accordingly, the RNAi agent used in the method of the present invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi double helix is ​​represented by formula (III): Justice: 5' n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3' Antonym: 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: i, j, k, and l are each independently 0 or 1; p, p', q and q' are each independently represented by 0 to 6; N a With N a Each 'sequence independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two different modified nucleotides; N b With N b 'It independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; in Every n p '、n p n q 'and n q The presence or absence of the dangling nucleotide is independent of the nucleotide itself; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif with three identical modifications on three consecutive nucleotides. In one state, 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 state, k is 0 and l is 0; or k is 1 and l is 0; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1. An exemplary combination of right and wrong stocks forming an RNAi double helix includes the following: 5' n p -N a -YY THE a -n q 3' 3' n p '-N a '-Y'Y'Y'-N a '-n q ' 5' (IIIa) 5' n p -N a -YY THE b -ZZ ZN a -n q 3' 3' n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a the q ‘ 5’ (IIIb) 5' n p -N a -XX XN b -YY THE 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 -XX XN b -YY YN b -ZZ ZN 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) When the RNAi agent is represented by formula (IIIa), each N a An oligonucleotide sequence is independently represented by 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. When the RNAi agent is represented by formula (IIIb), each N b Each N represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a An oligonucleotide sequence is independently represented by 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. When this RNAi formulation is represented as formula (IIIc), N b With N b Each 'N' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a An oligonucleotide sequence is independently represented by 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. When this RNAi formulation is represented as formula (IIId), N b With N b Each 'N' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a and N a ' is an oligonucleotide sequence that independently represents 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. N a N a '、N b and N b 'It is an independent modifier of alternating patterns. Each X, Y, and Z in equations (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same as or different from each other. When the RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one Y nucleotide can form a base pair with one Y' nucleotide. Alternatively, at least two Y nucleotides can form base pairs with their corresponding Y' nucleotides; or all three Y nucleotides can form base pairs with their corresponding Y' nucleotides. When the RNAi agent is represented by formula (IIIb) or (IIId), at least one Z nucleotide may form a base pair with one Z' nucleotide. Alternatively, at least two Z nucleotides may form base pairs with their corresponding Z' nucleotides; or all three Z nucleotides may form base pairs with their corresponding Z' nucleotides. When the RNAi agent is represented by formula (IIIc) or (IIId), at least one X nucleotide may form a base pair with one X' nucleotide. Alternatively, at least two X nucleotides may form base pairs with their corresponding X' nucleotides; or all three X nucleotides may form base pairs with their corresponding X' nucleotides. In a single-state sample, the modification on nucleotide Y is different from the modification on nucleotide Y', the modification on nucleotide Z is different from the modification on nucleotide Z', and / or the modification on nucleotide X is different from the modification on nucleotide X'. In a single-state sample, when the RNAi agent is represented by formula (IIId), N a The modification is either 2'-O-methyl or 2'-fluoro. In another state, when the RNAi agent is represented by formula (IIId), N a The modification system consists of 2'-O-methyl or 2'-fluoro modification, n p '>0, and at least one np' is linked to an adjacent nucleotide via a phosphate thioester linker. In another state, when the RNAi agent is represented by formula (IIId), N a The modification system consists of 2'-O-methyl or 2'-fluoro modification, n p '>0, and at least one n p The RNAi system is linked to adjacent nucleotides via phosphate thioester linkages, and the positive-positive strand is linked to one or more GalNAc derivatives via divalent and trivalent branched linkage groups. In another state, when the RNAi system is represented by formula (IIId), N a The modification system consists of 2'-O-methyl or 2'-fluoro modification, n p '>0, and at least one n p The 'is linked to an adjacent nucleotide via a thiophosphate linker, the positive nucleotide linker comprising at least one thiophosphate linker, and the positive nucleotide linker being associated with one or more GalNAc derivatives linked via divalent and trivalent branched linker groups. In a single-state sample, when the RNAi agent is represented by formula (IIIa), N a The modification system is 2'-O-methyl or 2'-fluoro, np'>0, and at least one n p The 'is linked to an adjacent nucleotide via a thiophosphate linker, the positive nucleotide linker comprising at least one thiophosphate linker, and the positive nucleotide linker being associated with one or more GalNAc derivatives linked via divalent and trivalent branched linker groups. In one state, the RNAi agent contains at least two double-helical polymers represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the double helices are linked by a linker. The linker may be cleavable or non-cleavable. If desired, the polymer may also contain a ligand. Each double helix may target the same gene or two different genes; or each double helix may target two different target sites of the same gene. In one state, the RNAi agent contains three, four, five, six, or more double-helical polymers represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the double helices are linked by a linker. The linker may be cleavable or non-cleavable. Depending on the desired effect, the polymer may also contain a ligand. Each double helix may target the same gene or two different genes; or each double helix may target two different target sites of the same gene. In one state, two RNAi agents, represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), are linked together at the 5' end and one or both 3' ends, and bind to a ligand as needed. Each of these agents targets the same gene or two different genes; or each of these agents targets two different target sites of the same gene. Multiple publications disclose polymeric RNAi agents that can be used in the methods of the present invention. These publications include World Patent No. 2007 / 091269, U.S. Patent No. 7,858,769, World Patent No. 2010 / 141511, World Patent No. 2007 / 117,686, World Patent No. 2009 / 014,887, and World Patent No. 2011 / 031520, the entire contents of which are incorporated herein by reference. RNAi agents containing one or more carbohydrate moieties that bind to an RNAi agent can optimize one or more properties of that RNAi agent. In many cases, the carbohydrate moieties are linked to a modified subunit of the RNAi agent. For example, the ribose of one or more ribonucleotides in a dsRNA agent can be replaced by other moieties, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is linked. In this document, a ribonucleotide subunit to which the ribose of the subunit has been replaced refers to a ribose substitution modification subunit (RRMS). The cyclic carrier can be a carbocyclic system, i.e., all ring atoms are carbon atoms; or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic system, or may contain two or more rings, such as a fused ring. The cyclic carrier can be a fully saturated cyclic system, or may contain one or more double bonds. The ligand can be linked to a polynucleotide via a carrier. Such carriers include (i) at least one "backbone linker," preferably two "backbone linkers," and (ii) at least one "tethering linker." Herein, a "backbone linker" refers to a functional group, such as a hydroxyl group, that can be used and is suitable for incorporating a carrier into a backbone, such as the backbone of a ribonucleic acid, or generally a bond, such as a phosphate ester or a modified phosphate ester, such as a sulfur-containing phosphate ester. In some cases, a "tethering linker" (TAP) refers to a building block ring atom of the cyclic carrier, such as a carbon atom or heteroatom (distinct from the atom providing the backbone linker) that links the selected portion of the system. The portion can be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Where desired, the selected portion of the system is linked to the cyclic carrier via spacer tethers. Therefore, the cyclic carrier often includes functional groups such as amines, or typically provides a bond that is suitable for incorporating or linking another chemical monolith, such as a ligand, to the building ring. The RNAi agent can be conjugated to a ligand via a carrier, wherein the carrier can be a cyclic or acyclic group; the cyclic group is preferably selected from pyrrolidyl, pyrazolinyl, pyrazolidyl, imidazolinyl, imidazolinyl, piperidinyl, and piperidinyl groups. base, [1,3] two Cyclopentyl, Azoxyl, isopropyl Azoxyl, morpholino, thiazolidinyl, isothiazolidinyl, quinolone linyl, pyridine Ketone, tetrahydrofuranyl, and decahydronaphthyl; the acyclic group is preferably selected from serine alcohol backbone or diethanolamine backbone. In certain specific embodiments, the RNAi agent used in the method of the present invention is 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 one listed in Table 12, the agent may lack a terminal dT. This invention further includes double-stranded RNAi agents comprising any of the sequences listed in any of Tables 1, 2, 4, 5, 8, 10, and 12, wherein the antisense strand contains a 5' phosphate ester or a phosphate ester mimic (see, for example, PCT Publication No. WO 2011005860). Furthermore, this invention includes double-stranded RNAi agents comprising any of the sequences listed in any of Tables 1, 2, 4, 5, 8, 10, and 12, wherein the antisense strand contains a 2' fluorine group replacing the 2'-OMe group at the 5' end. These agents may contain ligands. In one state, the agent is AD-60940 (positive stock: CfsusGfgUfaUfuUfCfCfuAfgGfgUfaCfaAfL96; negative stock: usUfsgUfaCfcCfuAfggaAfaUfaCfcAfgsasg). A. Ligands The double-stranded RNA (dsRNA) agent of this invention can bind to one or more ligands as needed. The ligand can be attached to the positive, negative, or both ends of the positive and negative strands at the 3', 5', or both ends. For example, the ligand can bind to the positive strand. In a preferred embodiment, the ligand binds to the 3' end of the positive strand. In a further preferred embodiment, the ligand is GalNAc. In a particularly preferred embodiment, the ligand is GalNAc. 3: In some states, the ligand, such as the GalNAc ligand, is attached to the 3' end of the RNAi agent. In one state, the RNAi agent binds to a ligand such as the GalNAc ligand, as shown in the figure below: Among them, X is O or S. In a single-state sample, X is O. Numerous entities can be coupled with the RNAi agent of this invention. Preferred components of the system are coupled directly or indirectly via septal chords. In a preferred state, the ligand system alters the distribution, targeting, or lifetime of the molecules it incorporates. In a preferred state, the ligand system provides enhanced affinity for selected targets such as molecules, cells or cell types, chambers, receptors such as cellular or organ chambers, tissues, organs, or body regions compared to those without the ligand. Ligands that provide enhanced affinity for selected targets are also called targeted ligands. Some ligands may possess endosomolytic properties. These endosomolytic ligand systems promote the lysis of endosomes and / or the transport of the components of the present invention or their components from the endosomes to the cytoplasm of the cell. The endosomolytic ligands may be polyanionic peptides or peptide-like compounds exhibiting pH-dependent membrane activity and fusogenicity. In one state, the endosomolytic ligand system is assumed to be in an active conformation at an endosomolytic pH. This "active" conformation is the conformation in which the endosomolytic ligand promotes the lysis of endosomes and / or the transport of the components of the present invention or their components from the endosomes to the cytoplasm of the cell. Exemplary cytocompatibility systems include GALA peptides (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), EALA peptides (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one state, the cytocompatibility component may contain chemical groups (e.g., amino acids) that will undergo charge or protonation changes in response to pH changes. The cytocompatibility component may be linear or branched. The ligands can improve the transport, hybridization, and specificity of the resulting natural or modified oligonucleotides, or polymer molecules containing any combination of monomers and / or natural or modified ribonucleotides disclosed herein, and can enhance nuclease resistance. Typically, ligands can include therapeutic modifiers, such as those used to enhance uptake; diagnostic compounds or reporter groups, such as those used to monitor distribution; cross-linking agents; and parts that impart resistance to nucleases. Common examples include lipids, steroids, vitamins, carbohydrates, proteins, peptides, polyamines, and peptide mimics. Ligands may include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulins); carbohydrates (e.g., polyglucan, polytricaglucan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, such as a synthetic polyamino acid, or an oligonucleotide (e.g., an aptamer). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropylene)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphates. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, pseudopeptide-polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides. Ligands may also include targeting bases, such as cell or tissue targets, such as lectins, glycoproteins, lipids, or proteins like antibodies, which bind to specific cell types such as kidney cells. Target bases may be thyroid-stimulating hormone, melanocyte-stimulating hormone, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetylglucosamine, N-acetylglucosamine, polymannose, polyfructose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, RGD peptides, RGD-like peptides, or aptamers. Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), and polycyclic aromatic hydrocarbons (e.g., phenazine). , dihydrophorine Artificial endonucleases or chelating agents (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acids, adamantine, 1-perylbutyric acid, dihydrotestosterone, 1,3-bis-O-hexadecylglycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleyl)lithocholic acid, O3-(oleyl)cholenic acid, dimethoxytriphenylmethyl, or phenazine) And peptide conjugates (e.g., tentacle peptides, Tat peptides), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG] 2. Polyamine, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraaza-macrocycle Eu3+ complexes), dinitrophenyl, HRP, or AP. Ligands can be proteins such as glycoproteins, or peptides such as molecules with specific affinity for coligands, or antibodies such as antibodies that bind to specific cell types such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylglucosamine, N-acetylglucosamine, polymannose, polyfructose, or aptamers. For example, the ligand can be lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The ligand can be a substance, such as a drug, that increases cellular uptake of iRNA agents. For example, this increase is achieved by disrupting the cellular cytoskeleton, such as by disrupting microtubules, microfilaments, and / or intermediate filaments. Such drugs can be, for example, taxon, vincristine, vinblastine, relaxin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. This ligand can increase cellular uptake of oligonucleotides by means of mechanisms such as activating inflammatory responses. Exemplary ligands with this effect include tumor necrosis factor-α (TNF-α), mesoleukin-1β, or gamma interferon. On one hand, the ligand is a lipid or lipid-based molecule. This lipid or lipid-based molecule preferably binds to serum proteins, such as human serum albumin (HSA). The ligand binding to HAS allows the conjugate to distribute to target tissues, such as non-renal target tissues of the body. For example, the target tissue could be the liver, including the parenchymal cells of the liver. Other molecules that can bind HAS can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid-based ligands can (a) increase the conjugate's resistance to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) can be used to modulate the binding to serum proteins such as HAS. Lipid-based ligands can be used to modulate, for example, the binding of the conjugate to the target tissue. For instance, lipids or lipid-based ligands that bind more strongly to the HAS are less likely to target the kidneys and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to the HAS can be used to make the conjugate target the kidneys. In a preferred embodiment, the lipid-based ligand binds to HAS. Preferably, it binds to HAS with an affinity sufficient to allow the conjugate to be preferentially distributed in non-renal tissues. However, it is preferable that the strength of this affinity is insufficient to cause irreversible binding of the HSA-ligand. In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HAS, thus the conjugate will be better distributed in the kidney. Alternatively, other fractions targeting kidney cells may be used instead of the lipid-based ligand, or the conjugate may contain other fractions in addition to the lipid-based ligand. On the other hand, this formulation utilizes components taken up by target cells, such as proliferating cells, such as vitamins. It is particularly useful for treating unwanted cell proliferation, such as malignant or non-malignant cell proliferation characterized by cancer cells. Exemplary vitamin systems include vitamins A, E, and K. Other exemplary vitamin systems include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins, or nutrients taken up by cancer cells. It also includes HAS, low-density lipoprotein (LDL), and high-density lipoprotein (HDL). On the other hand, the formulation is a cell penetrant, preferably a helical cell penetrant. The agent is preferably amphiphilic. An illustrative agent is a peptide, such as tat or antennopedia. If the agent is a peptide, it can be modified, including by peptide-like compounds, inverted isomers, non-peptide or pseudopeptide chains, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has both lipophilic and lipophobic phases. The ligand may be a peptide or a peptide mimic. A peptide mimic (also referred to herein as an oligopeptide mimic) is a molecule capable of folding into a three-dimensional structure similar to that of a natural peptide as defined. The length of the peptide or peptide mimic moiety is about 5 to 50 amino acids, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. The peptide or peptide mimic may be, for example, a cell-penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety may be a dendritic peptide, a restriction peptide, or a cross-linked peptide. In another alternative, the peptide moiety may include a hydrophobic membrane translocation sequence (MTS). An illustrative peptide containing a hydrophobic MTS is an RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) can also serve as targeting moieties. These peptide moieties can act as "delivery" peptides, capable of transporting polar macromolecules across the cell membrane, including peptides, oligonucleotides, and proteins. For example, sequences forming the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO: 13) and the Drosophila Antennapedia protein RQIKIWFQNRRMKWKK (SEQ ID NO: 14) have been found to function as delivery peptides. Peptides or peptide-like compounds can be encoded by random DNA sequences, such as those identified in phage-display libraries or one-bead-one-compound (OBOC) libraries (Lam et al., Nature, 354: 82-84, 1991). Preferably, the peptide or peptide mimic is attached to the iRNA agent via a combined monomeric unit, which is a cell-targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide or an RGD mimic. The length of the peptide moiety can range from about 5 amino acids to about 40 amino acids. These peptide moieties can have structural modifications to, for example, increase stability or target conformational properties. Any structural modifications disclosed below 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 promote the targeting of the iRNA agent to a variety of other tissues, including tumors of the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8: 783-787, 2001). Preferably, the RGD peptide will promote the targeting of the iRNA agent to the kidney.The RGD peptide can be linear or cyclic and can be modified, such as by glycosylation or methylation, to enhance targeting to specific tissues. For example, a glycosylated RGD peptide can deliver iRNA agents to tumor cells expressing α Vβ3 (Haubner et al., Jour. Nucl. Med., 42: 326-336, 2001). Peptides enriched in proliferating cells can be used as target markers. For example, RGD-containing peptides and peptide mimics can target cancer cells, particularly those exhibiting integrins. Therefore, RGD peptides, cyclic peptides containing RGD, RGD peptides including D-amino acids, and synthetic RGD mimics can be used. Besides RGD, other parts targeting this integrin ligand can be used. Typically, these ligands are used to control proliferating cells and angiogenesis. Preferred binding systems for this type of ligand target PECAM-1, VEGF, or other oncogenes such as those disclosed herein. "Cell-penetrating peptides" are peptides capable of penetrating cells, such as microbial cells (bacterial or fungal cells) or mammalian cells (human cells). Microbial cell-penetrating peptides can be, for example, α-helical 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 include nuclear localization signals (NLS). For example, cell-penetrating peptides can be amphipathic peptides composed of two parts, such as MPG, which is derived from the fusion peptide domain of the NLS of HIV-1 gp41 and the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31: 2717-2724, 2003). In a single-state sample, the targeting peptide can be an amphipathic α-helical peptide. Examples of amphipathic α-helical peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish gut antimicrobial peptides (HFIAPs), xenopus antimicrobial peptide, brevinins-2, dermaseptin, bee venom peptide, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin. It is preferred that a large number of factors maintain the overall stability of the helical structure. For example, the maximum number of helical stabilizing residues (e.g., leu, ala, or lys) and the minimum number of helical destabilizing residues (e.g., porphyrins or cyclic monomeric units) are used. The capping residue is considered to stabilize the helix; for example, Gly-series illustrative N-capping residues and / or C-terminal acetylation can be used to provide external hydrogen bonds. Stability can be provided by forming salt bridges between residues with opposite charges and separated by i±3 or i±4 positions. For example, cationic residues such as lysine, arginine, homoarginine, ornithine, or histamine can form salt bridges with anionic residues such as glutamate or aspartate. Peptides and peptide-like systems include those having naturally occurring or modified peptides, such as D or L peptides; α, β, or γ peptides; N-methyl peptides; azide peptides; peptides having one or more amides, i.e., peptides whose linkages are replaced by one or more urea, thiourea, carbamate, or sulfonylurea linkages; or cyclic peptides. The targeting ligand can be any ligand capable of targeting a specific receptor. Examples include: folic acid esters, GalNAc, galactose, mannose, mannose-6P, sugar clusters such as 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. These ligands can also be nucleic acids, such as aptamers. The aptamer can be modified, or have a combination of modifications disclosed herein. Intracellular release agents include imidazole, polyimide or oligoimide, PEI, peptide, gene fusion peptide, polycarboxylate, polycationic, masked oligo or polycationic or anionic, acetal, polyacetal, ketal / polyketal, orthoester, polymers with masked or unmasked cationic or anionic charges, and dendritic polymers with masked or unmasked cationic or anionic charges. PK modulators are representative pharmacokinetic modulators. PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, bile acids, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, neurolipids, naproxen, ibuprofen, vitamin E, biotin, etc. It is also believed that oligonucleotides containing a large number of thiophosphate chains bind to serum proteins; therefore, short oligonucleotides with multiple thiophosphate chains in their backbone, such as oligonucleotides having about 5, 10, 15, or 20 bases, are also considered ligands (e.g., as PK-regulating ligands) and are consistent with this invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) can also serve as PK regulatory ligands, which is consistent with the present invention. Other ligand binding systems conforming to the present invention are disclosed in U.S. Patent Nos. 10 / 916,185, filed August 10, 2004; 10 / 946,873, filed September 21, 2004; 10 / 833,934, filed August 3, 2007; 11 / 115,989, filed April 27, 2005; and 11 / 944,227, filed November 21, 2007, which are incorporated herein by reference in their entirety for all purposes. When two or more ligands are present, these ligands may all have the same properties, all have different properties, or some ligands may have the same properties while others have different properties. For example, ligands may have targeting properties, endosomolytic activity, or PK regulatory properties. In a preferred embodiment, these ligands may all have different properties. The ligand can couple to the oligonucleotide at multiple positions, for example, the 3'-end, 5'-end, and / or intermediate position. In a preferred embodiment, the ligand is bound to the oligonucleotide via a chalaza, as disclosed herein, with a carrier. The ligand, or a chalaza-linked ligand, may be present in the monomer when it is incorporated into the growing strand. In some embodiments, the ligand can be incorporated by coupling to the "precursor" monomer after it has been incorporated into the growing strand. For example, a monomer having a chalaza, such as TAP-(CH2)nNH2 (i.e., without the corresponding ligand), can be incorporated into the growing oligonucleotide strand. In subsequent operations, that is, after the precursor monomer is incorporated into the strand, the ligand can be subsequently coupled to the precursor monomer by coupling the electrophilic group of the ligand having an electrophilic group such as a pentafluorophenyl ester or an aldehyde group with the terminal nucleophilic group of the precursor monomer. In another example, monomers with chemical groups suitable for participating in the Click Chemistry reaction, such as azide or alkyne-terminated telomeres, can be incorporated. In subsequent operations, i.e., after incorporating the precursor monomer into the strand, ligands with complementary chemical groups, such as alkynes or azides, can be coupled to the precursor monomer by coupling the alkyne with the azide. For double-stranded oligonucleotides, the ligand can bind to one or both strands. In some states, the double-stranded iRNA formulation contains a ligand that binds to the sense strand. In other states, the double-stranded iRNA formulation contains a ligand that binds to the antisense strand. In some states, ligands can bind to nucleic acid bases, glycosomes, or riboside linkages in nucleic acid molecules. Binding to purine nucleic acid bases or their derivatives can occur at any position, including intra- and extra-cyclic atoms. In some states, the 2-, 6-, 7-, or 8-positions of purine nucleic acid bases bind to the conjugate. Binding to pyrimidine nucleic acid bases or their derivatives can also occur at any position. In some states, the 2-, 5-, and 6-positions of pyrimidine nucleic acid bases can be substituted by the conjugate. Binding to the glycosome of a nucleotide can occur at any carbon atom. Examples of carbon atoms in the glycosome that can bind to the conjugate include 2', 3', and 5' carbon atoms. The 1' position can also bind to the conjugate, such as a non-basic residue. Nucleotide linkages can also carry conjugates. For phosphorus-containing chains (e.g., phosphate diesters, thiophosphates, dithiophosphates, phosphonamides, etc.), the junction portion can directly bind to the phosphorus atom, or bind to the O, N, or S atom bonded to the phosphorus atom. For nucleotide chains containing amines or amides (e.g., PNA), the junction portion can bind to the nitrogen atom of the amine or amide, or bind to an adjacent carbon atom. Any suitable ligand in the field of RNA interference can be used, but the ligand is typically a carbohydrate, such as a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, or polysaccharide. The binding linkage of the ligand to the nucleic acid includes those described above. For example, the ligand may be one or more GalNAc (N-acetylglucosamine) derivatives bound via divalent and trivalent branched linkages. In one state, the dsRNA of the present invention binds to bivalent and trivalent branched strand junctions, including the structures shown in any of formulas (IV) to (VII): in: q 2A q 2B q 3A q 3B q 4A q 4B q 5A q 5B and q 5C Each occurrence of each number represents 0 to 20 independently, and the repeating units may be the same or different. P 2A P 2B P 3A P 3B P 4A P 4B P 5A P 5B P 5C T 2A T 2B T 3A T 3B T 4A T 4B T 4A T 5B T 5C Each occurrence is independent of the following: CO, NH, O, S, OC(O), NHC(O), CH. 2. CH 2NH or CH 2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each time it appears, it is independently absent; an enyl group, a substituted enyl group, wherein one or more methylene groups can be formed by O, S, S(O), SO. 2. One or more intervals or terminations of N(RN), C(R')=C(R"), C≡C or C(O); R 2A R 2B R 3A R 3B R 4A R 4B R 5A R 5B R 5C Each time it appears, it is considered independently non-existent: NH, O, S, CH. 2. C(O)O, C(O)NH, NHCH(R) a -C(O), -C(O)-CH(R) a )-NH-, CO, CH=NO, , , , , Or heterocyclic group; L 2A L 2B L 3A L 3B L 4A L 4B L 5A L 5B and L 5C The term "ligand" indicates that each instance of a ligand is independently a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R a It is a H or amino acid side chain. Trivalent GalNAc derivatives are particularly useful in combination with RNAi agents used to suppress the expression of target genes, such as those in formula (VII): Among them, L 5A L 5B and L 5C The series represents monosaccharides, such as GalNAc derivatives. Examples of suitable divalent and trivalent branched chain-bonded GalNAc derivatives include, but are not limited to, the following compounds: In other samples, the RNAi agent used in the method of this invention is AD-59743. III. Delivery of iRNA in this invention Delivery of the iRNA agent of the present invention to cells, such as those of a subject (e.g., human subjects with a need for it, such as those with TMPRSS6-related conditions like hemochromatosis), can be achieved through a variety of different pathways. For example, delivery can be performed by bringing cells into contact with the iRNA of the present invention, either in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition containing iRNA, such as dsRNA, to the subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors encoding and directing the expression of the iRNA. These alternatives are further discussed below. Typically, any method for delivering nucleic acid molecules (in vitro or in vivo) (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5): 139-144 and WO94 / 02595, both of which are incorporated herein by reference in their entirety) can be used in conjunction with the iRNA of this invention. For in vivo delivery, factors to be considered for the delivery of iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, for example, by direct injection or implantation into tissues or by topical administration. Local administration to the therapeutic site maximizes the local concentration of the agent, limits the agent’s exposure to systemic tissues that may be harmful to or degrade the agent, and allows for a lower total dose of the iRNA molecules to be administered. Several studies have shown successful knockout of gene products when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA, via intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24: 132-138) and subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9: 210-216), both showed prevention of angiogenesis in experimental models of age-related macular degeneration. Furthermore, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al (2005) Mol. Ther. 11: 267-274) and prolonged survival in 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 been shown to be successfully delivered locally to the CNS via direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thacker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602), and successfully delivered locally to the lungs via intranasal administration (Howard, KA., et al.). 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). For systemic administration of iRNA to treat diseases, the RNA can be modified or delivered using a drug delivery system; both methods work to prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutical carrier can also allow the iRNA components to target specific tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemically binding to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, systemic injection of iRNA targeting the ApoB binding to the lipophilic cholesterol moiety into mice resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al. (2004) Nature 432: 173-178). The binding of iRNA to its aptamer has been shown in a mouse model of prostate cancer to inhibit tumor growth and mediate tumor regression (McNamara, JO., et al. (2006) Nat. Biotechnol. 24: 1005-1015). Alternatively, the iRNA can be delivered using drug delivery systems such as nanoparticles, dendritic polymers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote binding to the negatively charged iRNA molecule and enhance interactions on the negatively charged cell membrane, allowing for efficient uptake of the iRNA by the cell.Cationic lipids, dendritic polymers, or polymers may be bonded to or induced to form carriers or microcells that encapsulate the iRNA (see, e.g., Kim SH., et al (2008) Journal of Controlled Release 129(2): 107-116). The formation of carriers or microcells further prevents the degradation of the iRNA when administered systemically. The methods for preparing and administering cationic iRNA complexes are entirely within the capabilities of those skilled in the art (see, e.g., 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, all of which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems that can be used for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), as described above; Verma, UN., et al (2003), as described above), Oligofectamine ("solid nucleic acid lipid particles") (Zimmermann, TS., et al (2006) Nature 441: 111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12: 321-328; Pal, A., et al (2005) Int J. Oncol. 26: 1087-1091), poly(ethylene imine) (Bonnet ME., 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 polyimide derivatives (Tomalia, DA., et al (2007) Biochem. Soc. Trans. 35: 61-67; Yoo, H., et al (1999) Pharm. Res. 16: 1799-1804). In some forms, iRNA forms a complex with cyclodextrin for systemic administration. Methods of administration of iRNA and cyclodextrin and pharmaceutical compositions can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety. A. Vector encoding the iRNA of this invention iRNAs targeting the TMPRSS6 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12: 5-10; Skillern, A, et al., PCT Application WO 00 / 22113; Conrad, PCT Application WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be short-term (on a timescale of hours to weeks) or long-term (on a timescale of weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, and can be integrative or non-integrative vectors. This transgene can also be constructed to be inherited as an extrachromosomal plastid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92: 1292). Individual or multiple strands of iRNA can be copied onto an expression vector by a promoter. If two independent strands are to be expressed to produce something such as dsRNA, the two independent expression vectors can be co-introduced (e.g., by transfection or infection) into the target cell. Alternatively, individual strands of dsRNA can be copied individually by a promoter, and both strands reside on the same expression plasmid. In one state, the dsRNA line appears as an inverted repeating polynucleotide linked by a linker polynucleotide sequence, thus possessing a stem and loop structure. Typically, iRNA expression vectors are DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant structures for the iRNA expression described herein. Eukaryotic cell expression vectors are well known in the art and are available from a variety of commercial sources. Typically, these vectors are provided with convenient restriction sites for inserting the desired nucleic acid fragment. The iRNA expression vector can be delivered systemically, such as by intravenous or intramuscular administration, by culturing target cells from the patient in vitro and subsequently reintroducing them into the patient, or by any other means of introducing the vector into the desired target cells. iRNA express plastids can be transfected into target cells as complexes with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid carriers (e.g., Transit-TKO™). This invention also focuses on various lipid transfection methods for targeting different regions of the target RNA for iRNA-mediated knockout over a period of one week or one month. Successful vector introduction into host cells can be monitored using various known methods. For example, short-term transfection can use reporter molecules such as fluorescent markers like green fluorescent protein (GFP). In vitro cell stability can be ensured by using markers (e.g., antibiotics and drugs) that provide transfected cells with resistance to specific environmental factors, such as hygromycin B resistance. Viral vector systems that can be used in conjunction with the methods and components disclosed herein include, but are not limited to, (a) adenovirus vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, monoclonal murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) piconemavirus vectors; (i) poxvirus vectors, such as orthopox virus vectors or avian poxvirus vectors such as fowlpox or canarypox virus vectors; and (j) helper-dependent or gutless adenovirus vectors. Replication-defective viruses may also be preferred. Different vectors may be incorporated into the genome of the cell, or not. If desired, these configurations may include viral sequences for transfection. Alternatively, this construct can be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Typically, constructs used for recombinant expression of iRNA require modulatory elements, such as promoters and boosters, to ensure the iRNA's expression in target cells. Other aspects of vectors and constructs are further revealed below. Vectors used for iRNA delivery will include regulatory elements (promoters, enhancers, etc.) sufficient to satisfy the desired iRNA expression in target cells or tissues. These regulatory elements can be selected to provide constitutive or modulated / inducible expression. For example, the expression of iRNA can be precisely modulated using inducible regulatory sequences sensitive to certain physiological modulators such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J.8: 20-24). Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, modulation using ecdysone, estrogen, progesterone, tetracycline, dimerizing chemical inducers, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in this art should be able to select appropriate regulatory / promoter sequences based on the desired use of iRNA transgenesis. Viral vectors containing nucleic acid sequences encoding iRNA can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors contain the components necessary for the proper packaging and integration of the viral genome into the host cell DNA. The nucleic acid sequence encoding iRNA is cloned into one or more vectors that facilitate the delivery of this nucleic acid to the patient. A more detailed description of retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6: 291-302 (1994), which reveals the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. Other references illustrative of the use of retroviral vectors in gene therapy include: Clowes et al., J. Clin. Invest. 93: 644-651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. In Genetics and Devel. 3: 110-114 (1993). Lentiviral vector systems intended for use include, for example, HIV-based vectors disclosed in U.S. Patents Nos. 6,143,520, 5,665,557, and 5,981,276, which are incorporated herein by reference. Adenoviruses are also anticipated to be used in the delivery of iRNA in this invention. Adenoviruses are highly attractive delivery vectors, such as for delivering genes to respiratory epithelial cells. Adenoviruses naturally infect respiratory epithelial cells, where they cause mild disease. Other targets of adenovirus delivery systems include the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the ability to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), describes gene therapy using adenovirus vectors. Bout et al., Human Gene Therapy 5:3-10 (1994) describes the use of adenovirus vectors to transfer genes into the respiratory epithelial cells of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in: Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication No. WO94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). AV vectors suitable for expressing the iRNA of the present invention, methods for constructing such recombinant AV vectors, and methods for delivering such vectors to target cells are disclosed in Xia H et al. (2002), Nat. Biotech. 20:1006-1010. Adeno-associated virus (AAV) vectors can also be used to deliver the iRNA of this invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993); U.S. Patent No. 5,436,146). In one state, the iRNA can be expressed as two independent, complementary single-stranded RNA molecules from a recombinant AAV vector having a promoter such as U6 or H1 RNA or a cellular giant virus (CMV) promoter. The AAV vectors suitable for expressing the dsRNA of the present invention, methods for constructing the recombinant AV vectors, and methods for delivering such vectors to target cells are disclosed in Samulski R et al. (1987), J.Virol. 61: 3096-3101; Fisher KJ et al. (1996), J.Virol, 70: 520-532; Samulski R et al. (1989), J.Virol. 63: 3822-3826; U.S. Patents Nos. 5,252,479 and 5,139,941; International Patent Application No. WO 94 / 13788; and International Patent Application No. WO 93 / 24641, the entire disclosure of which is incorporated herein by reference. Another viral vector system suitable for delivering the iRNA of the present invention is poxvirus, such as vaccinia virus, for example, attenuated poxvirus strains such as modified Ankara virus (MVA) or NYVAC, or avian poxviruses such as fowlpox or canarypox virus. Viral vector tropism can be pseudo-modeled by using envelope proteins or surface antigens from other viruses, or by modifying the vector by substituting different viral capsid proteins. For example, lentiviral vectors can be pseudo-modeled using surface proteins from vesicular stomatitis virus (VSV), rabies virus, Ebola virus, and Mokola virus. AAV vectors can be modified to express different capsid protein serotypes, thus making the vector target different cells; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference. The drug formulation of the vector may include a vector in an acceptable diluent, or may include a sustained-release matrix in which a gene delivery vector is embedded. Alternatively, if the complete gene delivery vector can be generated intact from recombinant cells, such as a retroviral vector, the drug formulation may include one or more cells that generate the gene delivery system. IV. Pharmaceutical composition of the present invention This invention also includes pharmaceutical compositions and formulations comprising the iRNAs of this invention. In one embodiment, what is provided herein is a pharmaceutical composition containing the iRNA disclosed herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions containing iRNA are intended for the treatment of diseases or conditions associated with TMPRSS6, such as hemochromatosis. These pharmaceutical compositions are formulated based on delivery modalities. One example is a composition formulated for systemic administration via non-enteric delivery, such as intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma via methods such as intracerebral infusion, such as continuous pump infusion. Pharmaceutical compositions comprising the RNAi agents of the present invention may be, for example, solutions with or without buffering, or compositions containing pharmaceutically acceptable carriers. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, microcellular formulations, emulsions, and gene therapy carriers. In the method of this invention, the RNAi agent can be administered in solution. Free RNAi agents can be administered in non-buffered solutions such as saline or water. Alternatively, monomeric siRNA can also be administered in a stable buffered solution. The buffered solution may contain acetate, citrate, prolyl, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate-buffered saline (PBS). The pH and osmotic pressure of the solution containing the RNAi agent can be adjusted to make it suitable for administration to the subject. In some formulations, the buffer solution further contains an agent for controlling the permeability of the solution, maintaining it at a desired value, such as the physiological value of human plasma. Solutes that can be added to the buffer solution to control its permeability include, but are not limited to, proteins, peptides, amino acids, non-metabolizing polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some formulations, the agent for controlling the permeability of the solution is a salt. In some formulations, the agent for controlling the permeability of the solution is sodium chloride or potassium chloride. The pharmaceutical composition of the present invention can be administered at a dose sufficient to inhibit the expression of the TMPRSS6 gene. Typically, the appropriate dose of the iRNA of the present invention can be in the range of about 0.001 to about 200.0 mg per kilogram of body weight per day, typically about 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered in single doses of about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 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. For example, this RNAi agent, such as dsRNA, can be administered in the following doses: approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3 1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges falling within these limits are also considered part of this invention. In another embodiment, the RNAi agent, such as dsRNA, is administered in the following doses: 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 / kg, 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 mg / kg. Approximately 50 mg / kg, approximately 20 to approximately 50 mg / kg, approximately 25 to approximately 50 mg / kg, approximately 25 to approximately 50 mg / kg, approximately 30 to approximately 50 mg / kg, approximately 35 to approximately 50 mg / kg, approximately 40 to approximately 50 mg / kg, approximately 45 to approximately 50 mg / kg, approximately 0.1 to approximately 45 mg / kg, approximately 0.25 to approximately 45 mg / kg, approximately 0.5 to approximately 45 mg / kg, approximately 0.75 to approximately 45 mg / kg, approximately 1 to approximately 45 mg / kg, approximately 1.5 to approximately 45 mg / kg, approximately 2 to approximately 45 mg / kg, approximately 2.5 to approximately 45 mg / kg, approximately 3 to approximately 45 mg / kg, approximately 3.5 to approximately 45 mg / kg, approximately 4 to approximately 45 mg / kg Approximately 4.5 to 45 mg / kg, approximately 5 to 45 mg / kg, approximately 7.5 to 45 mg / kg, approximately 10 to 45 mg / kg, approximately 15 to 45 mg / kg, approximately 20 to 45 mg / kg, approximately 20 to 45 mg / kg, approximately 20 to 45 mg / kg, approximately 25 to 45 mg / kg, approximately 25 to 45 mg / kg, approximately 30 to 45 mg / kg, approximately 35 to 45 mg / kg, approximately 40 to 45 mg / kg, approximately 0.1 to 40 mg / kg, approximately 0.25 to 40 mg / kg, approximately 0.5 to 40 mg / kg, approximately 0.75 to 40 mg / kg, approximately 1 to 40 mg / kg, approximately 1.5 to 40 mg / kb, approximately 2 to 40 mg / kg. 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 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 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 The values ​​are approximately 20 mg / kg, approximately 0.25 to approximately 20 mg / kg, approximately 0.5 to approximately 20 mg / kg, approximately 0.75 to approximately 20 mg / kg, approximately 1 to approximately 20 mg / kg, approximately 1.5 to approximately 20 mg / kg, approximately 2 to approximately 20 mg / kg, approximately 2.5 to approximately 20 mg / kg, approximately 3 to approximately 20 mg / kg, approximately 3.5 to approximately 20 mg / kg, approximately 4 to approximately 20 mg / kg, approximately 4.5 to approximately 20 mg / kg, approximately 5 to approximately 20 mg / kg, approximately 7.5 to approximately 20 mg / kg, approximately 10 to approximately 20 mg / kg, or approximately 15 to approximately 20 mg / kg. Values ​​and ranges falling within these limits are also considered part of this invention. For example, this RNAi agent, such as dsRNA, can be administered in the following doses: approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 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, or about 10 mg / kg. Values ​​and ranges within the specified limits are also considered part of this invention. In another embodiment, the RNAi agent, such as dsRNA, is administered in the following doses: about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg. g / 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 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg, 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 g, 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 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 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 / kg, about 1.5 to about 20 mg / kg, 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 state, the dsRNA was administered at a dose of approximately 10 mg / kg to approximately 30 mg / kg. Values ​​and ranges falling within these limits are also considered part of this invention. For example, therapeutic doses of iRNA can be administered to subjects, such as approximately 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 approximately 50 mg / kg. Values ​​and ranges falling within these limits are also considered part of this invention. In certain formulations, for example, when the composition of the present invention comprises the dsRNA disclosed herein and lipids, a therapeutic dose of iRNA can be administered to the subject, such as 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 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 The values ​​are approximately 5 mg / kg, 4.5 mg / kg, 4 mg / kg, 10 mg / kg, 5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, or 9.5 mg / kg to 10 mg / kg. Values ​​and ranges falling within these limits are also considered part of this invention. For example, the dsRNA can be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4. Administered at doses of 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, or approximately 10 mg / kg. Values ​​and ranges falling within the specified limits are also considered part of this invention. In certain embodiments of the present invention, for example, when a double-stranded RNAi agent comprises a modification (e.g., one or more motifs identically modified on three consecutive nucleotides, with one of these motifs included at or near the cleavage site of the agent), six phosphate-thioester chains, and a ligand, the agent is administered at doses of 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, or about 0 mg / kg. 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 Approximately 0.03 to approximately 0.4 mg / kg, approximately 0.03 to approximately 0.3 mg / kg, approximately 0.03 to approximately 0.2 mg / kg, approximately 0.03 to approximately 0.1 mg / kg, approximately 0.03 mg / kg to approximately 0.09 mg / kg, approximately 0.03 mg / kg to approximately 0.08 mg / kg, approximately 0.03 mg / kg to approximately 0.07 mg / kg, approximately 0.03 mg / kg to approximately 0.06 mg / kg, approximately 0.03 mg / kg to approximately 0.05 mg / kg, approximately 0.04 to approximately 0.5 mg / kg, approximately 0.04 to approximately 0.4 mg / kg, approximately 0.04 to approximately 0.3 mg / kg, approximately 0.04 to approximately 0.2 mg / kg, approximately 0.04 to approximately 0. Administered at doses of 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 mg / kg to about 0.5 mg / kg, about 0.05 mg / kg to about 0.4 mg / kg, about 0.05 mg / kg to about 0.3 mg / kg, about 0.05 mg / kg to about 0.2 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, about 0.05 mg / kg to about 0.09 mg / kg, about 0.05 mg / kg to about 0.08 mg / kg, or about 0.05 mg / kg to about 0.07 mg / kg.It is also considered that values ​​and ranges within the specified range are part of the present invention, for example, the RNAi agent can be administered to the subject at a dose of about 0.015 mg / kg to about 0.45 mg / mg. For example, the RNAi agent, such as the RNAi agent in a pharmaceutical composition, can be present in dosages of approximately 0.01 mg / kg, 0.0125 mg / kg, 0.015 mg / kg, 0.0175 mg / kg, 0.02 mg / kg, 0.0225 mg / kg, 0.025 mg / kg, 0.0275 mg / kg, 0.03 mg / kg, 0.0325 mg / kg, 0.035 mg / kg, 0... .0375mg / kg, 0.04mg / kg, 0.0425mg / kg, 0.045mg / kg, 0.0475mg / kg, 0.05mg / kg, 0.0525mg / kg ,0.055mg / kg, 0.0575mg / kg, 0.06mg / kg, 0.0625mg / kg, 0.065mg / kg, 0.0675mg / kg, 0.07mg / k g, 0.0725mg / kg, 0.075mg / kg, 0.0775mg / kg, 0.08mg / kg, 0.0825mg / kg, 0.085mg / kg, 0.0875 mg / kg, 0.09mg / kg, 0.0925mg / kg, 0.095mg / kg, 0.0975mg / kg, 0.1mg / kg, 0.125mg / kg, 0.15mg Dosages of 0.175 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, or about 0.5 mg / kg may be administered. Values ​​falling within the aforementioned limits are also considered part of this invention. The pharmaceutical composition can be administered once daily, or the iRNA can be administered at suitable intervals throughout the day as two, three, or more sub-dose, or even delivered by continuous infusion or through a controlled-release formulation. In this case, the amount of iRNA contained in each sub-dose must be correspondingly smaller to achieve the total daily dose. The dosage unit can also be formulated for delivery over several days, such as using conventional sustained-release formulations that provide continuous release of iRNA over a period of several days. Sustained-release formulations are known in the art and are particularly useful for delivering the agent at specific sites, such as those that can be used in combination with the agent of the present invention. In this configuration, the dosage unit contains a corresponding number of daily doses. In other embodiments, a single dose of the pharmaceutical composition may be long-acting, so subsequent doses may be administered at intervals not exceeding 3, 4, or 5 days, or at intervals not exceeding 1, 2, 3, or 4 weeks. In some embodiments of the present invention, a single dose of the pharmaceutical composition may be administered once weekly. In other embodiments of the present invention, a single dose of the pharmaceutical composition may be administered twice monthly. Those skilled in the art will recognize that certain factors can influence the dosage and timing required for effective treatment of subjects. These factors include, but are not limited to, the severity of the disease or condition, prior treatment, the subject's general health status and / or age, and any other pre-existing conditions. Furthermore, treatment of subjects with a therapeutically effective dose of the composition may include a single treatment or a series of treatments. The assessment of the effective dose and in vivo half-life of the individual iRNA covered by this invention can be performed using conventional methodologies or based on in vivo testing using suitable animal models, as disclosed elsewhere herein. The development of mouse genetics has led to the creation of numerous mouse models for studying various human diseases, such as iron overload-related conditions that would benefit from decreased TMPRSS6 expression. These models can be used for in vivo testing of iRNAs and for determining effective therapeutic doses. Suitable mouse models are known in the art and include, for example, Th3 / + thalassemia mice as a model of β-thalassemia (Douet et al., Am. J. Pathol. (2011), 178(2): 774-83); HFE knockout mice as a model of hereditary hemochromatosis (Zhou et al. (1998) Proc. Natl. Acad. Sci USA, 85: 2492-2497); and Uros (mut248) mice as a model of congenital erythropoietinosis (Ged et al. (2006) Genomics, 87(1): 84-92). The pharmaceutical composition of this invention can be administered via a variety of routes, depending on whether the desired treatment is local or systemic and the area to be treated. Administration can be topical (e.g., via transdermal patches), pulmonary administration such as inhalation or blowing in powder or aerosol, including via nebulizer; intratracheal administration, intranasal administration, epidermal administration, and transdermal administration; oral or non-enteral administration. Enteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal administration, such as via implanted devices; or intracranial administration, such as via intraparenchymal, intrathecal, or intracardiac administration. The iRNA can be delivered to a specific target tissue, such as the liver (e.g., hepatocytes). Pharmaceutical compositions and formulations for external application may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Traditional pharmaceutical carriers, aqueous, powdered, or oil-based bases, thickeners, etc., may be desired or required. Coated condoms, gloves, etc., may also be useful. Suitable external formulations include those described below, wherein the iRNA of the present invention is mixed with external delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and lipid systems include neutral (e.g., dioleoylphosphatidyl-DOPE ethanolamine, dimyristylphosphatidylcholine DMPC, distearate phosphatidylcholine), negative (e.g., dimyristylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropylDOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA of this invention can be encapsulated in liposomes, or can form complexes with the latter, especially cationic liposomes. Alternatively, the iRNA can be complexed with lipids, especially cationic lipids. Suitable fatty acids and their esters include, but are not limited to, arachidonic acid, oleic acid, arachidic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, perilla oleate, didecanoate, tridecanoate, monooleate, dilaurate, 1-monodecanoate, 1-dodecylazine-2-one, acetylcarnitine, acetylcholine, or C. 1-20 Alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are disclosed in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference. A. iRNA formulations containing membrane molecular components The iRNA used in the compositions and methods of this invention can be formulated for delivery into membrane molecular components, such as liposomes or microcells. Hereinafter, the term "liposome" refers to a vesicle composed of at least one bilayer, such as one or more bilayers, of amphipathic lipids. Lipid systems include monolayer and multilayer vesicles having a membrane formed from a lipophilic substance and an aqueous lumen. This aqueous portion contains the iRNA composition. The lipophilic substance separates the aqueous lumen from the aqueous exterior, although in some instances the aqueous exterior may include the iRNA composition, but typically it does not. Lipid systems are used to transfer and deliver active ingredients to a site of action. Because liposome membrane systems are structurally similar to biological membranes, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As the liposomes fuse with the cell, the internal aqueous contents, including the iRNA, are transported into the cell, where the iRNA can specifically bind to the target RNA and mediate RNAi. In some cases, liposomes can also specifically target, such as directing the iRNA to a specific cell type. Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent, thus forming microcells with the lipid component. For example, the lipid component can be an amphoteric cationic lipid or a lipid conjugate. The detergent can have a high critical microcell concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauryl sarcosine. Subsequently, an RNAi agent formulation is added to the microcells containing the lipid component. The cationic groups on the lipid react with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed by means such as dialysis to obtain the liposome formulation of the RNAi agent. If necessary, a carrier compound that facilitates the condensation reaction can be added during the reaction, for example, under controlled conditions. This carrier compound can be a polymer other than nucleic acids (e.g., spermine or spermidine). pH can also be adjusted to aid condensation. The method for manufacturing a stable polynucleotide delivery carrier, which incorporates a polynucleotide / cationic lipid complex as a structural component of the delivery carrier, is further disclosed in World Patent No. WO 96 / 37194, the entire contents of which are incorporated herein by reference. The formation of liposomes may also include one or more aspects of the illustrative methods disclosed in the following: Felgner, P.L. et al., Proc. Natl. Acad. Sci., USA 8: 7413-7417, 1987; U.S. Patents Nos. 4,897,355 and 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. Common techniques for preparing lipid aggregates of suitable size for use as delivery carriers include sonication and freeze-thaw extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). When a uniformly small (50 to 200 nm) and relatively homogeneous aggregate is desired, microfluidization can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). RNAi formulations can be readily encapsulated into liposomes using these methods. Lipid systems fall into two main categories. Cationic lipid systems consist of positively charged liposomes that react with negatively charged nucleic acid molecules to form stable complexes. These positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized within endosomes. Due to the acidic pH of these endosomes, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985). pH-sensitive or negatively charged lipid systems trap nucleic acids rather than misalign them. Since both nucleic acids and lipids are equally charged, repulsion occurs instead of misalignment. However, some nucleic acids trap within the aqueous lumen of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene into cell monolayers of cultures. The expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274). One major type of lipid composition includes phospholipids other than naturally derived phosphatidylcholine. Natural liposome compositions, for example, can be formed from dimyristylphosphatidylcholine (DMPC) or dipalmitophosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristylphosphatidylglycerol, while anionic membrane-fused lipid systems are primarily formed from dioleophosphatidylethanolamine (DOPE). Another type of lipid composition is formed from phosphatidylcholine (PC), such as, for example, soybean PC and egg PC. Yet another type is formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol. Examples of other methods for introducing liposomes in vitro and into cells in vivo include U.S. Patents Nos. 5,283,185 and 5,171,678; World Patents Nos. WO 94 / 00569, WO 93 / 24640 and WO 91 / 16024; Felgner, J. Biol. Chem. 269: 2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90: 11307, 1993; Nabel, Human Gene Ther. 3: 649, 1992; Gershon, Biochem. 32: 7143, 1993; and Strauss EMBO J. 11: 417, 1992. Nonionic liposome systems have also been examined to determine their applicability in drug delivery to the skin, particularly in systems containing nonionic surfactants and cholesterol. (Novasome is mentioned as an example.) TM I (Dilaurate / Cholesterol / Polyoxyethyl-10-stearyl ether) and Novasome TMA nonionic lipid formulation of glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether was used to deliver cyclosporine-A to the dermis of mouse skin. Results showed that this nonionic liposome system was effective in promoting the deposition of cyclosporine A in different layers of the skin (Hu et al. STPPharma.Sci., 1994, 4(6) 466). Liposomes also include "stereostabilized" liposomes, which, in this context, refers to liposomes containing one or more specific lipids, resulting in a longer cycle life compared to those lacking such specific lipids when these specific lipids are incorporated into the liposome. Examples of stereostabilized liposomes are those in which a portion of the vesicle-forming lipid site (A) of the liposome contains one or more glycolipids, such as monosialoganglioside G. M1 (B) is derived from one or more hydrophilic polymers such as polyethylene glycol (PEG). Although not wishing to be bound by any particular theory, this art holds that, at least for stereostabilized liposomes containing gangliosides, sphingomyelin, or PEG-derived lipids, the increased circulating half-life of such stereostabilized liposomes is due to reduced uptake by reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765). Various lipid systems comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported the ability of monosialotetrazolium ganglioside GM1, galactocerebroside sulfate, and phosphatidylinositol to improve the blood half-life of liposomes. These findings were described by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and World Patent No. WO 88 / 04924, granted to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) ganglioside G. M1 Or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. A lipid system containing 1,2-sn-dimyristylphosphatidylcholine is disclosed in World Patent No. WO 97 / 13499 (Lim et al.). In this study, cationic liposomes were used. Cationic lipid systems have the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although they cannot fuse effectively with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages. Other advantages of liposomes include: biocompatibility and biodegradability of lipid systems derived from natural phospholipids; the ability to encapsulate a wide range of water-soluble and lipid-soluble drugs; and the protection of 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). In the preparation of liposome formulations, important considerations include the lipid surface charge, vesicle size, and water volume of these liposomes. Positively charged synthetic cationic lipids, such as N-[1-(2,3-dioleoxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously react with nucleic acids to form lipid-nucleic acid complexes. These complexes can fuse with negatively charged lipids in tissue culture cells to deliver RNAi agents (see, for example, Felgner, PLE et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355, illustrating DOTMA and its use in combination with DNA). The DOTMA analog, 1,2-bis(oleoxy)-3-(trimethylamine)propane (DOTAP), can be used in combination with phospholipids to form DNA-widgetoids. Lipofectin TM (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells containing positively charged DOTMA liposomes that spontaneously contact negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the resulting complexes also carry a positive net charge. The positively charged complexes prepared in this way spontaneously bind to the negatively charged cell surface, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into cells such as tissue cultures. Another commercially available cationic lipid, 1,2-bis(oleyloxy)-3,3-(trimethylamine)propane (DOTAP) (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleyl group is linked by an ester rather than an ether. Other reported cationic lipid compounds include those that have been conjugated to multiple moieties, including, for example, carboxyspermyl, which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermyl glycine dioctadecanylamine (DOGS). TM Promega, Madison, Wisconsin) and Dipalmitoylphosphatidylethanolamine 5-carboxy-spermine-acetylamine (DPPES) (see, for example, U.S. Patent No. 5,171,678). Another cationic lipid conjugation system includes lipid derivatization with cholesterol (DC-Chol), which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179: 280, 1991). It has been reported that lipid polylysine conjugated to DOPE is effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065: 8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more effective 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 lipid systems suitable for delivering oligonucleotides are disclosed in WO 98 / 39359 and WO 96 / 37194. Lipid formulations are particularly suitable for topical administration, offering several advantages over other formulations. These advantages include reducing the side effects of high systemic absorption of the administered drug, increasing the accumulation of the administered drug at the desired target, and enhancing the ability to deliver RNAi agents into the skin. In some practical applications, lipid systems are used to deliver RNAi agents to epidermal cells and to enhance the penetration of RNAi agents into epidermal tissues such as the skin. For example, liposomes can be applied topically. It has been documented that drugs formulated as liposomes are delivered topically to the skin (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. Meth. Enz. 149: 157-176, 1987; Straubinger, R.M. and Papahadjopoulos, D. Meth. Enz. 101: 512-527, 1983; Wang, C.C. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987). Nonionic liposome systems have also been examined to determine their applicability in drug delivery to the skin, particularly in systems containing nonionic surfactants and cholesterol. 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 drugs to the dermis of mouse skin. These RNAi-containing formulations are used to treat skin conditions. Liposomes, including those containing iRNA, can be made highly deformable. This deformability allows the liposomes to penetrate through pores smaller than their average radius. For example, transferrin systems are a type of deformable liposome. Transferrins can be made by adding surface edge activators, typically surfactants, to a standard lipid composition. Transferrins containing RNAi agents can be delivered via methods such as subcutaneous infusion, thereby delivering the RNAi agent to keratinocytes in the skin. To traverse intact mammalian skin, lipid vesicles must pass through a series of pores, each with a diameter less than 50 nm, under the influence of an appropriate transdermal gradient. Furthermore, due to the properties of lipids, these transferrins can be self-optimized (adapting to the shape of pores in the skin), self-repairing, often reaching their target without segmentation, and frequently self-loading. Other formulations conforming to this invention are disclosed in U.S. Provisional Applications No. 61 / 018,616 (filed January 2, 2008), No. 61 / 018,611 (filed January 2, 2008), No. 61 / 039,748 (filed March 26, 2008), No. 61 / 047,087 (filed April 22, 2008), and No. 61 / 051,528 (filed May 8, 2008). PCT Application No. PCT / US2007 / 080331 (filed October 3, 2007) also discloses formulations following this invention. Another type of liposome, the transferor system, is a highly deformable lipid aggregate, making it an attractive candidate for drug delivery carriers. Transferors can be described as lipid droplets that are highly deformable, allowing them to easily penetrate through pores smaller than the droplet itself. Transferors are adaptable to their application environment; for example, they are self-optimizing (adapting to the shape of pores in the skin), self-repairing, frequently reaching their target without segmentation, and often self-loading. To create transferors, surface edge activators, typically surfactants, can be added to a standard lipid composition. Transferors have been used to deliver serum albumin to the skin. It has been shown that transferor-mediated serum albumin delivery is as effective as subcutaneous injection of a solution containing serum albumin. Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of various types of surfactants (both natural and synthetic) is by hydrophilic / lipophilic balance (HLB). The natural properties of the hydrophilic group (also known as the "head") provide the most useful means of classifying the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285). If a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical and cosmetic products and can be used over a wide pH range. Typically, based on their structure, their HLB values ​​range from 2 to approximately 18. Nonionic surfactants include nonionic esters, such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, desiccant esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamines and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this category. Polyoxyethylated surfactants are the most common members of the nonionic surfactant class. If a surfactant molecule carries a negative charge when dissolved or dispersed in water, it is classified as anionic. Anionic surfactants include carboxylates, such as soaps; acetylated lactates; amino acid acetylated amides; sulfuric acid esters, such as alkyl sulfates and ethoxylated alkyl sulfates; sulfonates, such as alkylbenzene sulfonates, acetylated hydroxyethyl sulfonates, acetylated taurines and sulfosuccinates; and phosphate esters. The most important members of the anionic surfactant class are alkyl sulfates and soaps. If a surfactant molecule carries a positive charge when dissolved or dispersed in water, it is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. These quaternary ammonium salts are the most commonly used members of this class. If a surfactant molecule has the ability to carry either a positive or negative charge, then the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids. A review of the use of surfactants in pharmaceutical products, formulations and emulsions has been conducted (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285). The iRNA used in the method of this invention can also be provided as a microcell formulation. Hereinafter, "microcell" is defined as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure, such that all the hydrophobic portions of these molecules face inwards, leaving the hydrophilic portions in contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists. Suitable for transdermal membrane delivery of stable mixed microcellular formulations, which can be achieved by using an aqueous solution of the siRNA component and an alkali metal C 8 to C 22The microcells are prepared by mixing alkyl sulfates and cell-forming compounds. Exemplary cell-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, perilla oleate, glyceryl monooleate, monooleate esters, monolaurate esters, borage oil, evening primrose oil, menthol, trihydroxyoxycholic acid and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, trioleyl glycerol, polyoxyethyl ethers and their analogues, polidocanol alkyl ethers and their analogues, chenodeoxycholate, deoxycholate, and mixtures thereof. These cell-forming compounds may be added simultaneously with or after the alkali metal alkyl sulfate. The mixed microcells utilize substantially any mixture of these components, excluding vigorous mixing, to provide microcells of smaller size. In one method, a first microcellular composition is prepared containing the siRNA composition and at least the alkali metal alkyl sulfate. Subsequently, the first microcellular composition is mixed with at least three microcellular-forming compounds to form a mixed microcellular composition. In another method, the microcellular composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulfate, and at least one microcellular-forming compound, followed by adding the remaining microcellular-forming compound under vigorous mixing. Phenol and / or m-cresol can be added to the mixed microcellular composition to stabilize the formulation and protect it from bacterial growth. Alternatively, phenol and / or m-cresol can be added together with the microcellular components. After the mixed microcellular composition is formed, isotonic agents such as glycerol can also be added. To deliver the microcellular formulation as a spray, the formulation is placed in an aerosol disperser, which is then filled with propellant. The propellant in the disperser is in liquid form under pressure. The ratio of these components is adjusted so that the aqueous phase and the propellant become one phase; that is, one phase exists. If two phases exist, the disperser must be agitated before dispersing a portion of the contents, such as through a metering valve. The dosage of the formulation is propelled from the metering valve in the form of a fine spray. Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorinated carbons, dimethyl ethers, and diethyl ethers. In certain formulations, HFA 134a (1,1,1,2-tetrafluoroethane) may be used. The specific concentration of the main components can be determined by relatively simple experiments. For absorption through the oral cavity, the dosage administered via gastrointestinal injection or administration is often increased by at least two or three times. B. Lipid particles The iRNA of the present invention, such as dsRNA, can be completely encapsulated in lipid formulations such as LNP or other nucleic acid-lipid particles. In this document, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs are highly suitable for systemic application due to their extended circulation life after intravenous (iv) injection and accumulation at distal sites (e.g., sites physically separated from the administration site). LNPs include "pSPLP," which comprises encapsulated concentrate-nucleic acid complexes, as detailed in PCT Publication WO 00 / 03683. The particles of this invention typically have an average diameter 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. Furthermore, when present in the nucleic acid-lipid particles of this invention, these nucleic acids are resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and their preparation methods are disclosed in U.S. Patents Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410 and 6,815,432, U.S. Publication No. 2010 / 0324120, and PCT Publication No. WO 96 / 40964. In a single sample, the ratio (mass / mass ratio) of the lipid to the drug (e.g., the ratio of lipid to dsRNA) will be in the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges falling within the above-defined ranges are also considered part of this invention. This cationic lipid can be, for example, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide, etc. Dimethylammonium (DDAB), N-(I-(2,3-dioleoxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleoxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoxy)propylamine (DODMA), 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleoaminomethoxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoxy-3-(dimethylamino)ethyl Acryloxypropane (DLin-DAC), 1,2-dilinolenic acid-3-N-morpholinylpropane (DLin-MA), 1,2-dilinolenic acid-3-dimethylaminopropane (DLinDAP), 1,2-dilinolenic acid-3-dimethylaminopropane (DLin-S-DMA), 1-linolenic acid-2-linolenic acid-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinolenic acid-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinolenic acid-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinolenic acid-3-(N-methylpiperyl)propane (DLin-TAP.Cl) 1,2-Dilinoleylamino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dilinoleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxane (DLin-K-DMA) (3aR,5s,6aS)-N,N-dimethyl-2,2-bis((9Z,12Z)-octadecane-9,12-dienyl)tetrahydro-3aH-cyclopentane[d][1,3]dioxane-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperyl) -1-yl)ethylazinediyl)di-dodecane-2-ol (Tech G1), or a mixture thereof. The cationic lipids may contain about 20 mol% to about 50 mol% or about 40 mol% of the total lipids present in the particles. In another embodiment, compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxane can be used to prepare lipid-siRNA nanoparticles. 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxane is disclosed in U.S. Provisional Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference. In one state, the lipid-siRNA particles consist of 40% 2,2-dilinole-4-dimethylaminoethyl-[1,3]-dioxane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (molar percentage), with a particle size of 63.0 ± 20 nm and an siRNA / lipid ratio of 0.027. The ionizable / non-cationic lipid can be anionic or neutral, including, but not limited to, distearate phosphatidylcholesterol (DSPC), dioleoylphosphatidylcholesterol (DOPC), dipalmitoylphosphatidylcholesterol (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoleoylphosphatidylcholesterol (POPC), and palmitoleoylphosphatidylethanolamine. (POPE), dioleinylphosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitinylphosphatidylethanolamine (DPPE), dimyristinylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleinylphosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Based on the total lipids present in the particles, the non-cationic lipids, if including cholesterol, may be approximately 5 mol% to approximately 90 mol%, approximately 10 mol%, or approximately 58 mol%. The conjugated lipids that inhibit particle aggregation can be, for example, polyethylene glycol (PEG) lipids, including, but not limited to, PEG-diacetylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-dilauoxypropyl (C12), PEG-dimyristoxypropyl (C14), PEG-dispalmitoxypropyl (C16), or PEG-distearateoxypropyl (C18). The conjugated lipids that prevent particle gelation can be from 0 mol% to about 20 mol% or about 2 mol% based on the total lipids present in the particles. In some samples, the nucleic acid-lipid complex includes cholesterol, which is approximately 10 mol% to approximately 60 mol% or approximately 48 mol% based on the total lipids present in the particle. In a single-state sample, lipid-based ND98-4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). The respective feedstock solutions in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-Ceramide C16, 100 mg / ml. Subsequently, the ND98, cholesterol, and PEG-Ceramide C16 feedstock solutions can be combined in a molar ratio such as 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in a sodium acetate solution at pH 5), resulting in a final ethanol concentration of approximately 35% to 45% and a final sodium acetate concentration of approximately 100 to 300 mM. The lipid-dsRNA nanoparticles typically form spontaneously during mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., with a 100 nm cutoff), for example, using a hot extruder such as the Lipex Extruder (Northern Lipids, Inc.). In some cases, this extrusion step can be avoided. Ethanol removal and simultaneous buffer exchange can be performed, for example, by dialysis or tangential flow filtration. The buffer can be exchanged via, for example, phosphate-buffered saline (PBS) at approximately pH 7, such as approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. The LNP01 formulation is disclosed in, for example, International Patent Application No. WO 2008 / 042973, which is incorporated herein by reference. Other illustrative lipid-dsRNA formulations are disclosed in Table A. Table A Formulations containing LNP (1,2-dilinoleoxy-N,N-dimethylaminopropane (DLinDMA)) are disclosed in International Patent No. WO2009 / 127060, filed on April 15, 2009, which is incorporated herein by reference. Formulations containing XTC are disclosed in U.S. Provisional Application No. 61 / 148,366, filed January 29, 2009; U.S. Provisional Application No. 61 / 156,851, filed March 2, 2009; U.S. Provisional Application No. 61 / 228,373, filed July 24, 2009; U.S. Provisional Application No. 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference. Formulations containing MC3 are disclosed in, for example, U.S. Patent Publication No. 2010 / 0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference. Formulations containing ALNY-100 are disclosed in, for example, International Patent Application No. PCT / US09 / 63933, filed on November 10, 2009, which is incorporated herein by reference. The formulation containing C12-200 is disclosed in U.S. Provisional Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference. Synthesis of ionizable / cationic lipids Any compound 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 detailed in the examples. Unless otherwise specified, all substituent systems are as defined below. "Alkyl" refers to saturated aliphatic hydrocarbons containing 1 to 24 carbon atoms, either straight-chain or branched, and in acyclic or cyclic form. Representative saturated straight-chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; while saturated branched-chain alkyl groups include isopropyl, dibutyl, isobutyl, tributyl, and isopentyl. Representative saturated cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; while unsaturated cyclic alkyl groups include cyclopentenyl and cyclohexenyl. "Alkenyl" refers to an alkyl group containing at least one double bond between adjacent carbon atoms. Alkenyl groups include both cis and trans isomers. Representative straight-chain and branched-chain alkenyl groups include vinyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, etc. "Alynyl" refers to an alkyl or alkenyl group that additionally contains at least one triple bond between adjacent carbon atoms. Representative straight-chain and branched-chain alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, etc. "Acryl" refers to an alkyl, alkenyl, or alkynyl group whose carbon atom at the bonding site is substituted with a side oxygen group, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl groups are acryl groups. "Heterocycle" refers to a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic ring, which is saturated, unsaturated, or aromatic, containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the nitrogen and sulfur heteroatoms may be oxidized as needed, and the nitrogen heteroatom may be quaternized as needed. "Heterocycle" also includes any of the above-mentioned heterocycles fused to a benzene ring in a bicyclic ring. Heterocycles may be bonded via heteroatoms or carbon atoms. Heterocyclic systems include heteroaryl groups as defined below. Heterocyclic systems include morpholino, pyrrolidone, pyrrolylalkyl, piperidinyl, and piperinyl groups. It includes compounds such as hydroxyl, hydantoin, valproic acid, ethylene oxide, oxetane, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiophenyl, tetrahydrothiophenyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiophenyl. The terms "alkyl group to be substituted as desired", "alkenyl group to be substituted as desired", "alkynyl group to be substituted as desired", "acetylated group to be substituted as desired", and "heterocyclic group to be substituted as desired" mean that, when substituted, at least one hydrogen atom is replaced by a substituent. In the example of a side oxygen substituent (=O), two hydrogen atoms are replaced. In this regard, the substituent system includes oxy groups, halogens, heterocycles, -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, Rx and Ry are the same or different, and are independently hydrogen, alkyl, or heterocycles, and the alkyl and heterocycle substituents may each be further substituted by one or more of the oxy group, halogen, -OH, -CN, alkyl, -ORx, heterocycle, -NRxRy, -NRxC(=O)Ry, -NRxSO2Ry, -C(=O)Rx, -C(=O)ORx, -C(=O)NRxRy, -SOnRx, and -SOnNRxRy. "Halogen" refers to fluorine, chlorine, bromine, and iodine. In some states, the method of the present invention may require the use of a protecting group. Protecting group methodology is well known to those skilled in the art (see, for example, *Protective Groups in Organic Synthesis*, Green, T. Wet et al., Wiley-Interscience, New York City, 1999). In short, a protecting group as used herein is any group that reduces or eliminates the undesirable reactivity of a functional group. A protecting group may be added to a functional group to mask its reactivity in certain reactions, and then removed to expose the original functional group. In some states, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the undesirable reactivity of an alcohol functional group. Protecting groups may be added and removed using techniques known in the art. Synthesis of Formula A In certain samples, the nucleic acid-lipid particles of the present invention are formulated using cationic lipids of formula A: Among them, R 1 With R 2 It is an independent alkyl, alkenyl, or alkynyl group, each of which may be substituted as needed; and R 3 With R 4 It is independently a lower alkyl group, or R 3 With R 4They can be combined to form heterocycles that are substituted as needed. In some cases, the cationic lipid is XTC (2,2-dilinoleno-4-dimethylaminoethyl-[1,3]-dioxane). Typically, the lipid of formula A above can be prepared by reaction formula 1 or 2 below, wherein, unless otherwise specified, all substituents are as defined above. Reaction 1 Lipid A can be prepared according to reaction formula 1, wherein R 1 With R 2 It can be alkyl, alkenyl or ynyl, each of which may be substituted as needed; and R 3 With R 4 It is independently a lower alkyl group, or R 3 With R 4 They can be combined to form heterocyclic rings that require substitution. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. The reaction of 1 and 2 yields ketal 3. Ketone 3 is treated with amine 4 to obtain lipids of formula A. Lipids of formula A can be converted to the corresponding ammonium salts by treatment with organic salts of formula 5, wherein X is selected from antagonistic anions such as halogens, hydroxides, phosphates, and sulfates. Reaction 2 Alternatively, the starting material ketone 1 can be prepared according to reaction formula 2. Green's reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. The reaction of 6 and 7 yields ketone 1. The conversion of ketone 1 to the lipid of corresponding formula A is as disclosed in reaction formula 1. MC3 Synthesis The preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate) is as follows. A solution of (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid, followed by a dilute aqueous sodium bicarbonate solution. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed using a rotary evaporator. The residue was eluted through a silica column (20 g) using a gradient of 1 to 5% methanol / dichloromethane eluent. The fractions containing the purified product were combined and the solvent was removed to obtain a colorless oil (0.54 g). Synthesis of ALNY-100 The synthesis of ketal 519 [ALNY-100] was carried out using the following reaction formula 3: Reaction 3 Synthesis of 515 In a two-necked RBF (1 L), under a nitrogen atmosphere at 0 °C, a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF was slowly added to LiAlH2O with stirring. 4 (3.74 g, 0.09852 mol) was added to a suspension in 200 mL of anhydrous THF. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. The reaction progress was monitored by TLC. After the reaction was complete (by TLC), the mixture was cooled to 0 °C, and saturated Na was carefully added. 2SO The reaction was quenched by a 4-solution. The reaction mixture was stirred at room temperature for 4 hours and then filtered. The residue was thoroughly washed with THF. The filtrate and washings were mixed and distilled with 400 mL of diethylcarbamate. The alkane was diluted with 26 mL of concentrated HCl and stirred at room temperature for 20 minutes. Volatile substances were removed under vacuum to provide 515 hydrochloride as a white solid. Yield: 7.12 g. 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). Synthesis of 516 In 250 mL of two-necked RBF, NEt3 (37.2 mL, 0.2669 mol) was added to a solution of compound 515 in 100 mL of dry DCM with stirring. The mixture was cooled to 0 °C under a nitrogen atmosphere. Then, N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) dissolved in 50 mL of dry DCM was slowly added, and the mixture was allowed to warm to room temperature. After the reaction was complete (2 to 3 hours, monitored by TLC), it was continuously added with 1 N HCl solution (1 × 100 mL) and saturated NaHCO3. The mixture was washed with 3 solutions (1 × 50 mL). The organic layer was then rinsed with anhydrous Na₂O. 2SO 4. The product was dried, and the solvent was evaporated to obtain the crude product. Purification by silica gel column chromatography yielded 516 as a viscous substance. Yield: 11 g (89%). ¹H-NMR (CDCl₃, 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%). Synthesis of 517A and 517B At room temperature, in a 500 mL RBF container, cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of acetone and water (10:1). N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was then added, followed by 4.2 mL of OsO2. 4 (0.275 g, 0.00108 mol) was added to a 7.6% solution of tributanol. After the reaction was complete (approximately 3 hours), solid Na was added. 2SO 3. Purify the mixture and stir it at room temperature for 1.5 hours. Dilute the reaction mixture with DCM (300 mL), and then successively with water (2 × 100 mL) and saturated NaHCO3. Wash with 3 (1×50 mL) solution, water (1×30 mL), and finally brine (1×50 mL). The organic phase was rinsed with anhydrous Na₂SO₄. 2SO 4. Drying and removing solvent under vacuum. The crude product was purified by silica gel column chromatography to obtain a mixture of non-mirror image isomers, which were separated by preparative HPLC. Yield: Approximately 6 g of crude product. 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 was confirmed by X-ray diffraction. Synthesis of 518 Compound 518 (1.2 g, 41%) was obtained as a colorless oil using a process similar to that used to synthesize compound 505. ¹H-NMR (CDCl₃, 400 MHz): δ = 7.35–7.33 (m, 4H), 7.30–7.27 (m, 1H), 5.37–5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58–4.57 (m, 2H), 2.78–2.74 (m, 7H), 2.06–2.00 (m, 8H), 1.96–1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37–1.25 (br m, 36H), 0.87 (m, 6H). HPLC: 98.65%. The usual process for synthesizing compound 519 A solution of compound 518 (1 eq) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40 °C for 0.5 hours, followed by further cooling in an ice bath. The solution was then saturated with Na... 2SO The mixture was carefully hydrolyzed with an aqueous solution, then filtered through diatomaceous earth and reduced to an oil. Column chromatography analysis provided a pure 519 (1.3 g, 68%) as a colorless oil. 13C NMR δ=130.2,130.1(x2),127.9(x3),112.3,79.3,64.4,44.7,38.3,35.4,31.5,29.9(x2),29.7,29.6(x2),29.5(x3),29.3(x2),27.2(x3),25.6,24.5,23.3,226,14.1; Electrolytic MS (+ve): C44H80NO2(M+H)+ The calculated molecular weight was 654.6, and the measured value was 654.6. Formulations prepared using standard or non-extrusion methods can be characterized using similar methods. For example, formulations are typically characterized by visual inspection. They should be whitish, translucent solutions free of aggregates or precipitates. The particle size and size distribution of lipid nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). The particle size should be approximately 20 to 300 nm, such as 40 to 100 nm. The particle size distribution should be unimodal. The total dsRNA concentration and entrapped fraction in the formulation are assessed using a dye exclusion assay. Prepared dsRNA samples can be incubated with RNA-binding dyes such as Ribogreen (molecular probes) in the presence or absence of interfering surfactants such as 0.5% Triton-X100. The total dsRNA in this formulation can be determined relative to a standard curve using a signal from a sample containing the surfactant. The trapping fraction is determined by subtracting the "free" dsRNA content (measured by a signal in the absence of the surfactant) from the total dsRNA content. The percentage of trapped dsRNA is typically >85%. For LNP formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Suitable ranges are typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm. Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, pouches, tablets, or small tablets. Thickeners, flavorings, diluents, emulsifiers, dispersants, or binders may be desired. In some cases, the oral formulation is one in which the dsRNA of the present invention is administered in combination with one or more penetration-enhancing surfactants and chelating agents. Suitable surfactants include fatty acids and / or their esters or salts, bile acids and / or their salts. Suitable bile acid / salt systems include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, deoxyglycholic acid, taurocholic acid, deoxytaurocholic acid, sodium tauro-24,25 dihydrofusidate, and sodium glycodihydrofusidate. Suitable fatty acid families include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, decanoic acid, myristic acid, palmitic acid, stearic acid, linolenic acid, perilla oleate, didecanoate, tridecanoate, monooleic glyceride, dilauric glyceride, 1-monodecanoic glyceride, 1-dodecylazine-2-hepta-one, acetylcarnitine, acetylcholine, or their monoglycerides, diglycerides, or pharmaceutically acceptable salts (e.g., sodium salts). In some formulations, combinations of penetration enhancers are used, for example, combinations of fatty acid / salts and bile acid / salts. An exemplary combination is sodium salt of lauric acid, decanoic acid, and UDCA. Other penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA of this invention can be orally delivered in particulate form, including spray-dried particulates or a combination of microparticles or nanoparticles. DsRNA miscible agents include polyamino acids; polyimides; polyacrylates; alkyl polyacrylates; polyoxethanes; alkyl cyanoacrylates; cationic gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylates; DEAE-derived polyimides, pollulan, cellulose and starch.Suitable chelating agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistamine, polyseminamine, protamine, polyvinylpyridine, polythiodiethylaminomethylvinylpyridine (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-dextrin, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and methods for their preparation are disclosed in detail in U.S. Patent No. 6,887,906, U.S. Publication No. 20030027780, and U.S. Patent No. 6,747,014, which are incorporated herein by reference in their respective cases. Compositions intended for administration outside the intestines, intracerebral parenchyma (to the brain), intrathecal, intracardiac, or intrahepatic administration may include sterile aqueous solutions and may also contain buffers, diluents, and other suitable adjuvants such as, but not limited to, penetrants, carrier compounds, and other pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions of this invention include, but are not limited to, solutions, emulsions, and lipid-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, pre-formed liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases such as liver cancer, liver-targeting formulations are particularly preferred. The pharmaceutical formulations of this invention can be conveniently presented in a single dosage form and can be prepared using conventional techniques known in the pharmaceutical industry. These techniques include the step of combining the active ingredient with a drug carrier or excipient. Typically, these formulations are prepared by uniformly and tightly binding the active ingredient with a liquid carrier or finely segmented solid carrier, or both, and then, if desired, shaping it into a product. The components of this invention can be formulated into one of a variety of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The components of this 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, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextrin. The suspension may also contain stabilizers. C. Additional preparations lotion The components of this invention can be prepared and formulated as emulsions. An emulsion is typically a heterogeneous system in which one liquid is dispersed in another liquid as droplets with a diameter greater than 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, NY, Volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker). Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Generally, an emulsion is a two-phase system comprising two closely mixed and immiscible liquid phases dispersed within each other. Emulsions are typically in the form of water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely fractionated and dispersed as small droplets in a large volume of oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely fractionated and dispersed as small droplets in a large volume of aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion. In addition to the dispersed phase and the active pharmaceutical ingredient, which can exist as a solution in the aqueous phase, oil phase, or as an independent phase, an emulsion may also contain additional components. If necessary, pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion. Pharmaceutical emulsions can also be multi-phase emulsions, consisting of more than two phases, such as, for example, oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions.These complex formulations often offer certain advantages that simple two-phase emulsions do not possess. For example, multi-emulsion systems where individual oil droplets encapsulate small water droplets within an o / w emulsion construct w / o / w emulsions. Similarly, systems where water droplets encapsulated within oil droplets are stabilized in an oily continuous phase provide o / w / o emulsions. Emulsions are characterized by very low or no thermodynamic stability. The dispersed or discontinuous phase of an emulsion is often well dispersed in an added or continuous phase and maintained in this form by means of emulsifiers or the viscosity of the formulation. Each phase of the emulsion can be semi-solid or solid, as exemplified by emulsion-type ointment bases and creams. Other methods of stabilizing emulsions require the use of emulsifiers that can be incorporated into any phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorbent matrices, and finely dispersed solids (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Synthetic surfactants, also known as surfactants, have been found to have wide applications in emulsion formation, as reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199). Surfactants are typically amphiphilic and contain both hydrophilic and hydrophobic components. The ratio of hydrophilic to hydrophobic properties of a surfactant is called the hydrophilic / lipophilic balance (HLB), and it is a valuable tool for classifying and selecting surfactants in formulation preparation. Surfactants can be classified into different categories based on their hydrophilic groups: nonionic, anionic, cationic, and amphoteric (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285). Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and gum arabic. Absorbent matrices possess hydrophilic properties, allowing them to saponify water to form w / o emulsions while maintaining their semi-solid consistency, such as anhydrous lanolin and hydrophilic paraffin oil. Finely fractionated solids have also been used as good emulsifiers, particularly in combination with surfactants in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swellable clays such as bentonite, palygorskite, pyroxene, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate. Many non-emulsifying substances can also be included between emulsions and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Hydrophilic colloidal or hydrocolloid systems include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., gum arabic, agar, alginate, carrageenan, guar gum, ark gum, and tragacanth gum), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers). These are dispersed or swollen in water to form a colloidal solution, which stabilizes the emulsion by a strong interfacial film surrounding the dispersed phase droplets and by increasing the viscosity of the added phase. Because emulsions often contain a large number of components, such as carbohydrates, proteins, sterols, and phospholipids that readily support microbial growth, these formulations often incorporate preservatives. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of parabens, and boric acid. Antioxidants are also often added to emulsion formulations to prevent spoilage. The antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisoles, and butylated hydroxytoluene; reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin. The application of emulsion formulations via the skin, oral and non-enteric routes, as well as their manufacturing methods, have been 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; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Due to their ease of formulation and efficacy from the perspective of absorption and bioavailability, emulsion formulations for oral delivery have been widely used (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional preparations are commonly used as oral emulsions. ii. Microemulsion In one embodiment of the present invention, the iRNA and nucleic acid components are formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and an amphiphilic compound, which is a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Typically, a microemulsion is prepared by first dispersing an oil in an aqueous solution of a surfactant, followed by the addition of a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a transparent system. Therefore, microemulsions have also been revealed as thermodynamically stable, isotropic, and clear dispersions 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, pages 185-215). Microemulsions are generally prepared by combining three to five components, including oil, water, surfactants, co-surfactants, and electrolytes. Whether the microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the characteristics of the oil and surfactants used, as well as the structure and geometry of the polar heads and hydrocarbon tails of these surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271). The phenomenological approach using phase diagrams has been extensively studied and has provided those familiar with the technique with comprehensive knowledge of how to formulate microemulsions (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335). Compared to traditional emulsions, microemulsions offer the following advantages: they allow water-insoluble drugs to dissolve in formulations that spontaneously form thermodynamically stable droplets. Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaglycerate (PO500), decaglycerol monodecanoate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), and decaglycerol decaoleate (DAO750), used alone or in combination with co-surfactants. These co-surfactants are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, which increase interfacial fluidity by penetrating the surfactant film and creating disordered films due to the voids between surfactant molecules. However, microemulsions can be prepared without co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in this art. The aqueous phase can typically include, but is not limited to, water, aqueous solutions of pharmaceuticals, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, substances such as Captex 300; Captex 355; Capmul MCM; fatty acid esters; medium-chain (C8-C12) mono, di, and triglycerides; polyoxyethylated fatty acid glycerides; fatty alcohols; PEGylated glycerides; saturated PEGylated C8-C10 glycerides; vegetable oils; and silicone oils. From the perspective of drug solubility and enhanced drug absorption, microemulsions are of particular interest. 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. Patents Nos. 6,191,105, 7,063,860, 7,070,802, and 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the following advantages: improved drug solubility, protection against enzymatic hydrolysis, potentially enhanced drug absorption due to surfactant-induced changes in membrane fluidity and penetration, ease of preparation, ease of oral administration in solid dosage forms, improved clinical potential, and reduced toxicity (see, for example, U.S. Patents Nos. 6,191,105, 7,063,860, 7,070,802, and 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Typically, microemulsions spontaneously form when the components are aggregated at room temperature. This is particularly advantageous when formulating heat-labile drugs, peptides, or iRNAs. In cosmetic and pharmaceutical applications, microemulsions have been used to effectively deliver active ingredients transdermally. It is anticipated that the microemulsion composition and formulation of the present invention will promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improve local cellular uptake of iRNA and nucleic acids. The microemulsions of this invention may also contain additional components and adjuvants 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 this invention. Penetration enhancers used in the microemulsions of this invention can be classified into one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating, non-surfactant agents (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these types has been disclosed above. iii. Particles The RNAi agent of this invention can be incorporated into particles such as microparticles. Microparticles can be prepared by spray drying, but can also be prepared by other methods, including freeze drying, evaporation, fluidized bed drying, vacuum drying, or combinations thereof. iv. Insert lifting agent In this invention, various penetration enhancers are employed to influence the efficient delivery of nucleic acids, particularly iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipophilic or lipid-soluble drugs readily cross cell membranes. It has been found that by treating the membrane to be penetrated with a penetration enhancer, even non-lipophilic drugs can cross the cell membrane. In addition to facilitating the diffusion of non-lipophilic drugs across the cell membrane, penetration enhancers also improve the penetration ability of lipophilic drugs. Penetration enhancers can be categorized into one of five broad groups: surfactants, fatty acids, bile salts, chelating agents, and non-chelating, non-surfactant agents (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 these types of penetration enhancers is described in more detail below. Surfactants (or "surfactants") are chemical entities that, when dissolved in an aqueous solution, reduce the surface tension of that solution or the interfacial tension between the aqueous solution and another liquid, thereby enhancing the absorption of iRNA through mucous membranes. Besides bile salts and fatty acids, these penetration enhancers include, for example, sodium dodecyl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorinated chemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252). Many fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, decanoic acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, perilla oleate, didecanoate, tridecanoate, monooleic glycerol (1-monooleyl-rac-glycerol), dilaurate, caprylic acid, arachidonic acid, 1-monodecanoate, 1-dodecylazine-2-one, acetylcarnitine, acetylcholine, their C1-20 alkyl esters (e.g., methyl ester, isopropyl ester, and tributyl ester), and their monoglycerides and diglycerides (e.g., oleate, laurate, decanoate, myristicate, palmitate, stearate, linoleic acid ester, 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). The physiological role of bile includes promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Many natural bile salts and their synthetic derivatives act as penetration enhancers. Therefore, the term "bile salts" includes any naturally occurring bile components and any synthetic derivatives thereof.Suitable bile salt systems include, for example, cholic acids (or their pharmaceutically acceptable sodium salts, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glutamic acid (sodium glutamic acid), glycocholic acid (sodium glycocholate), deoxyglycocholic acid (sodium deoxyglycocholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fumonisin (STDHF), sodium glycodihydrofumonisin, and polyoxyethylene-9-lauryl ether (POE) (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee 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. al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583). Chelating agents, when used in conjunction with the present invention, can be defined as compounds that remove metal ions from solution by forming complexes with them, thereby enhancing the absorption of iRNA through mucosa. Considering that they are used as penetration enhancers in the present invention, and since most characteristic DNA nuclease systems require divalent metal ions for catalysis and are therefore inhibited by chelating agents, chelating agents also have the addition advantage of being 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-acetylated derivatives of collagen, polyethylene glycol monolaurate (laureth-9), and N-aminoacetylated 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). In this article, non-chelating, non-surfactant penetration-enhancing compounds can be defined as compounds that exhibit insignificant activity as chelating agents or surfactants, but still enhance the absorption of iRNA through the digestive tract mucosa (see, for example, Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Such penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazine-ketone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626). Agents that enhance cellular iRNA uptake can also be incorporated into the pharmaceutical and other components of this invention. For example, cationic lipids such as lipofectin (Junichi et al., U.S. Patent No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysin (Lollo et al., World Patent No. WO 97 / 30731) are known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for instance, lipofectin. TM (Invitrogen; Carlsbad, CA), Lipofectamine 2000 TM (Invitrogen; Carlsbad, CA), 293fectin TM (Invitrogen; Carlsbad, CA), Cellfectin TM (Invitrogen; Carlsbad, CA), DMRIE-C TM (Invitrogen; Carlsbad, CA), FreeStyle TMMAX (Invitrogen; Carlsbad, CA), Lipofectamine TM 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine TM (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine TM (Invitrogen; Carlsbad, CA), Optifect TM (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 transfection reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP liposome transfection reagent (Grenzacherstrasse, Switzerland), DOSPER liposome transfection reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam ® Reagents (Promega; Madison, WI), TransFast TM Transfection reagents (Promega; Madison, WI), Tfx TM -20 reagent (Promega; Madison, WI), Tfx TM -50 reagent (Promega; Madison, WI), DreamFect TM (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass a D1 transfection reagent (New England Biolabs; Ipswich, MA, USA), LyoVec TM / LipoGen TM (Invitrogen; San Diego, CA, USA), PerFectin transfection reagent (Genlantis; San Diego, CA, USA), NeuroPORTER transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin transfection reagent (Genlantis; San Diego, CA, USA), BaculoPORTER transfection reagent (Genlantis; San Diego, CA, USA), TroganPORTER TM Transfection reagent (Genlantis; San 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 TM (B-Bridge International, Mountain View, CA, USA) etc. Other agents may be used to enhance the penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones, and terpenes such as limonene and menthone. v. carrier Some components of this invention also incorporate a carrier compound in the formulation. Hereinafter, "carrier compound" or "carrier" may refer to a nucleic acid or its analogue that is inert (i.e., not biologically active itself) but identified as a nucleic acid by in vivo processes, thereby reducing the bioavailability of the biologically active nucleic acid by means of, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. Co-administration of nucleic acids with a carrier compound, typically in an overdose of the latter, can lead to a substantial reduction in the amount of nucleic acid recovered from the liver, kidneys, or other external circulation organs, presumably due to competition between the carrier compound and the nucleic acid for common receptors. For example, when co-administered with polyinosinic acid, dextrin sulfate, polycytidic acid, or 4-acetaminophen-4'-isothiocyanate-zirconia-2,2'-disulfonic acid, the partial thiophosphorylated dsRNA recovered from liver tissue may be reduced (Miyao et al., DsRNA Res.Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183). vi. excipients In contrast to carrier compounds, "pharmaceutical carriers" or "excipients" are pharmaceutically acceptable solutions, suspensions, or any other pharmacologically inert carriers used to deliver one or more nucleic acids to animals. When used in combination with nucleic acids and other components of a given drug composition, the excipient may be liquid or solid, and the choice is made taking into account the dosing protocol to provide the desired volume, consistency, etc. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium glycolate); and wetting agents (e.g., sodium lauryl sulfate). The compositions of this invention may also be formulated using pharmaceutically acceptable organic or inorganic excipients that do not react adversely with nucleic acids and are suitable for non-enteral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc. Topical formulations of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oil bases. These solutions may also contain buffers, diluents, and other suitable adjuvants. Pharmaceutically acceptable organic or inorganic excipients suitable for non-enteric administration that do not react adversely with nucleic acids may be used. Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc. vii. Other components The compositions of this invention may additionally contain other additive components conventionally found in pharmaceutical compositions, in amounts consistent with the levels established in this art. Thus, for example, such compositions may contain additional compatible pharmaceutically active substances, such as, for instance, antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional substances suitable for the physical formulation of various dosage forms of the compositions of this invention, such as dyes, fragrances, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when such substances are added, they should not excessively interfere with the composition of the compositions of this invention. If necessary, such formulations may be sterilized and mixed with excipients that do not adversely react with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, fragrances, and / or aromatic substances. Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextrin. The suspension may also contain stabilizers. In some embodiments, the pharmaceutical composition of the present invention comprises (a) one or more iRNA compounds, and (b) one or more agents that act via non-RNAi mechanisms and are used to treat hemophilia. Examples of such agents include, but are not limited to, anti-inflammatory agents, anti-steatodendrostatic agents, antiviral agents, and / or anti-fibrotic agents. Furthermore, other substances commonly used to protect the liver, such as silymarin, may also be used in combination with the iRNAs disclosed herein. Other agents for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir, and others disclosed in U.S. Patent Applications Nos. 2005 / 0148548, 2004 / 0167116, and 2003 / 0144217 by Tung et al., and U.S. Patent Application No. 2004 / 0127488 by Hale et al. The toxicity and efficacy of these compounds can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, such as those used to determine LD50 (the dose that is 50% lethal to the population) and ED50 (the dose that is 50% effective in treating the population). The dose-to-efficacy ratio is the therapeutic coefficient, which can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a high therapeutic coefficient are preferred. Data obtained from cell culture assays and animal studies can be used to determine the range of human dosages. The dosages of the components of the invention described herein are generally within a range of circulating concentrations including those with low or no toxicity (ED50). This dosage may vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of the invention, the therapeutically effective dosage can be estimated from the initial cell culture assay. Doses can be determined in animal models to achieve a range of circulating plasma concentrations of the compound or, where appropriate, a range of plasma concentrations of the target sequence polypeptide product (e.g., achieving a reduced polypeptide concentration), which includes the IC50 measured in cell culture. 50 (That is, the concentration of the test compound that achieves 50% maximum inhibition of the symptoms). This information can be used to more accurately determine the useful dose for humans. Plasma levels can be measured, for example, by high-performance liquid chromatography. In addition to the above-described administration methods, the iRNA of this invention can be administered in combination with other known agents that are effective in treating lesions mediated by iron overload and treatable by inhibiting TMPRSS6 expression. In any case, the attending physician can adjust the dosage and schedule of the iRNA based on the results observed using standard efficacy assays known in the art or disclosed herein. V. Methods to suppress TMPRSS6 expression This invention provides a method for inhibiting the expression of TMPRSS6 (protein lyase-2) in cells. The method involves contacting cells with an RNAi agent, such as a double-stranded RNAi agent, in an amount sufficient to inhibit the expression of TMPRSS6 in the cells, thereby inhibiting the expression of TMPRSS6 in the cells. Cells can be contacted with a double-stranded RNAi agent either in vitro or in vivo. In vivo contact with the RNAi agent includes contacting cells or cell groups from a subject, such as a human subject, with the RNAi agent. Combinations of in vitro and in vivo contact methods are also possible. As mentioned above, contact can be direct or indirect. Furthermore, cell contact can be implemented via targeted ligands, including any ligands disclosed herein or known in the art. In a preferred embodiment, the targeted ligand contains a carbohydrate fraction, 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 the subject. In this article, the term "inhibition" is used interchangeably with "reduction," "silence," "adjustment," and other similar terms, and includes inhibition at any level. The phrase "suppression of TMPRSS6 gene expression" refers to the suppression of the expression of any TMPRSS6 gene (e.g., mouse TMPRSS6 gene, rat TMPRSS6 gene, monkey TMPRSS6 gene, or human TMPRSS6 gene) and its variants or mutants. Therefore, in this article, the TMPRSS6 gene in the cells, cell groups, or organisms where gene manipulation is performed can be the wild-type TMPRSS6 gene, the mutant TMPRSS6 gene, or the transgenic TMPRSS6 gene. "Suppression of TMPRSS6 gene expression" includes suppression of the TMPRSS6 gene at any level, such as at least partial suppression of TMPRSS6 gene expression. TMPRSS6 gene expression can be assessed based on levels, changes, or any variations in levels associated with TMPRSS6 gene expression, such as TMPRSS6 mRNA levels, TMPRSS6 protein levels, or lipid levels. These levels can be assessed at the individual cell or cell population level, including, for example, samples derived from subjects. The inhibition can be assessed by a reduction in the absolute or relative level of one or more variables associated with TMPRSS6 performance compared to a control level. The control level can be any type of control level used in this art, such as a baseline level before administration, or a level measured from subjects, cells, or samples that are untreated or treated with a control (e.g., a control of the buffer-only drug or a non-active agent control). In certain embodiments of the method of the present invention, the expression of the TMPRSS6 gene is suppressed 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%. The suppression of TMPRSS6 gene expression can be demonstrated by a decrease in the amount of mRNA expressed in a first cell or cell group (such cells may be present, for example, in a sample derived from a subject), in which the TMPRSS6 gene is transcribed and treated (e.g., by exposing the cells to the RNAi agent of the present invention, or by administering the RNAi agent of the present invention to a subject in whom such cells are present or were previously present). Therefore, compared to a second cell or cell group (target cells) that is substantially the same as the first cell or cell group but not treated in this way, the expression of the TMPRSS6 gene is suppressed. In a preferred embodiment, the suppression is assessed by expressing the level of mRNA in the treated cells as a percentage of the mRNA in the control cells using the following formula: Alternatively, suppression of TMPRSS6 gene expression can be assessed by a decrease in parameters that functionally link to TMPRSS6 gene expression, such as TMPRSS6 protein expression in tissues or serum, hepcidin gene or protein expression, or iron levels. TMPRSS6 gene silencing can be determined in cells expressing TMPRSS6 using any assay known in the art, either constructively or through genetic engineering. The liver is a major site of TMPRSS6 expression. Other significant sites of expression include the kidneys and uterus. Inhibition of TMPRSS6 protein expression can be demonstrated by a decrease in the level of TMPRSS6 protein expressed in cells or cell populations (e.g., in samples derived from subjects). As explained above for assessments of mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations. Control cell lines or cell populations that can be used to evaluate the inhibition of TMPRSS6 gene expression include cells or cell populations that have not yet been exposed to the RNAi agent of the present invention. For example, the control cells or cell populations may be derived from individual subjects (e.g., human or animal subjects) before treatment with the RNAi agent. The level of TMPRSS6 mRNA expressed in cells or cell populations can be determined using any method known in this art for assessing mRNA expression. In a single-state sample, the level of TMPRSS6 expression is determined by detecting transcribed polynucleotides or portions thereof, such as the mRNA of the TMPRSS6 gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assays using ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12: 7035), northern blotting, in situ hybridization, and microarray analysis. In a single-state sample, the level of TMPRSS6 expression was determined using a nucleic acid probe. In this document, the term "probe" refers to any molecule capable of selectively binding to a specific TMPRSS6. Probes may be synthesized by a person skilled in the art or derived from suitable biological agents. Probes may be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. The isolated mRNA can be used for hybridization or amplification assays, including, but not limited to, Southern blotting or Northern blotting analysis, polymerase chain reaction (PCR) analysis, and probe assays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) capable of hybridizing to TMPRSS6 mRNA. In one state, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as a nitrocellulose membrane. In another state, the probe is immobilized on a solid surface and contacted with the mRNA, for example, in Affymetrix gene chip assays. A skilled technician can easily modify known mRNA detection methods to determine TMPRSS6 mRNA levels. Another method for determining the level of TMPRSS6 expression in a sample includes RT-PCR (experimental phenotypes detailed in Mullis, 1987, US Pat. No. 4, 683, 202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193), self-persistent sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional 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 (Lizardi et al. The process involves amplification of mRNA and / or reverse transcriptase (to prepare cDNA) in a sample using methods such as al. (US Pat. No. 5, 854, 033) or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques known to those skilled in the art. These detection strategies are particularly useful for detecting nucleic acid molecules present in very low numbers. In a particular aspect of this invention, the level of TMPRSS6 is determined by quantitative fluorescent RT-PCR (i.e., the TaqMan™ system). The expression levels of TMPRSS6 mRNA can be monitored using membrane blotting (such as Northern Blot, Southern Blot, Dot Blot, etc.) or microwells, sample tubes, gels, microbeads, or fibers (or any solid support containing bonded nucleic acids). See U.S. Patents Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. The determination of TMPRSS6 expression levels can also be performed using nucleic acid probes in solution. In preferred embodiments, branched-strand DNA (bDNA) assays or real-time PCR (qPCR) are used to assess mRNA expression levels. The use of these methods is disclosed and illustrated in the examples herein. The level of TMPRSS6 protein expression can be determined using any method known in this art for measuring protein levels. These methods include, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, liquid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays, and electrochemiluminescence immunoassays. In this document, the term "sample" refers to an aggregate of similar fluids, cells, or tissues isolated from a subject, as well as an aggregate of fluids, cells, or tissues present within a subject's body. Examples of biological fluids include blood, serum and serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, and eye discharge. Tissue samples may include samples from tissues, organs, or localized areas. For example, a sample may originate from a specific organ, a portion of an organ, or fluids or cells within such organs. In some forms of samples, the sample may originate from the liver (e.g., the whole liver, or certain sections of the liver, or certain types of cells in the liver, such as hepatocytes). In a preferred form, "sample derived from a subject" refers to blood or plasma drawn from that subject. In yet another form, "sample derived from a subject" refers to liver tissue derived from that subject. In some embodiments of the method of the present invention, the RNAi agent is administered to a subject, thereby delivering the RNAi agent to a specific site within the subject's body. Inhibition of TMPRSS6 expression can be assessed using changes in the levels or levels of TMPRSS6 mRNA or TMPRSS6 protein in a fluid or tissue sample derived from the specific site within the subject's body. In a preferred embodiment, the site is the liver. The site may also be a subgroup or subset of cells from any of the aforementioned sites. The site may also include cells expressing a specific type of receptor. VI. Methods for treating or preventing TMPRSS6-related conditions This invention also provides methods for treating or preventing diseases and symptoms that can be regulated by the expression of the TMPRSS6 gene. For example, the compositions disclosed herein can be used to treat any condition associated with iron overload, such as thalassemia (e.g., β-thalassemia or α-thalassemia), primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, erythropoietinosis, sideroblastic anemia, hemolytic anemia, erythropoiesis-dysplastic anemia, or sickle cell anemia. In one embodiment, the TMPRSS6 iRNA is used to treat heme disorders. The TMPRSS6 iRNA of this invention can also be used to treat elevated iron levels due to other symptoms such as chronic alcoholism. In thalassemia, the bone marrow synthesizes insufficient amounts of heme chains; this subsequently reduces red blood cell production and causes anemia. It can affect either the alpha or beta chains, but is more common in beta-thalassemia. Newborns are healthy because their bodies still produce HbF, which lacks the beta chain; however, in the first few months of life, the bone marrow switches to producing HbA, and symptoms begin to appear. β-thalassemia originates from mutations in the non-expressive (β°) or low-expressive (β+) pair of the HBB gene. The severity of β-thalassemia depends on the genotype and includes mild / phenotypic β-thalassemia (β / β° or β / β+), intermediate β-thalassemia (β° / β+), and severe β-thalassemia (β° / β° or β° / β+). Intermediate thalassemia (TI) typically involves very little hemolysis, while severe β-thalassemia (TM) typically involves excessive hemolysis, leading to conditions such as anemia and splenomegaly; and highly inefficient erythropoiesis, resulting in bone marrow drive (skeletal changes, osteoporosis), increased erythropoietin synthesis, hepatosplenomegaly, hemoglobin depletion (megablastic anemia), and high uric acid levels in the blood. The iRNAs of this invention, such as TMPRSS6 iRNA, are better suited for treating iron overload in typical thalassemia with more TI-like symptoms (e.g., for treating individuals with β° / β+, β / β°, or β / β+ genotypes). Symptoms of β-thalassemia also include, for example, endocrine disorders, liver fibrosis, and myocardial fibrosis caused by complications of treatment such as iron overload. Administration of iRNA agents targeting TMPRSS6 can effectively treat one or more of these symptoms. Alpha-thalassemia originates from mutations in the efficacious (a°) or hypoefficacious (a+) pairs of the HBA1 or HBA2 genes. The severity of alpha-thalassemia depends on the genotype and includes phenotypic thalassemia (-α / α α), Hb Bart and Hydrops fetalis (a° / a°), mild alpha-thalassemia (- / α α), (-α / -α), and HbH disease (- / -a). It produces low-grade alpha-globulin chains, leading to an excess of β-chains in adults and an excess of γ-chains in newborns. The excess β-chains form unstable tetramers (called 4β-chain heme H or HbH), which have abnormal oxygen dissociation profiles. Administration of iRNA agents targeting TMPRSS6 is effective in treating iron overload in subjects with alpha-thalassemia. Symptoms of hemochromatosis include, for example, abdominal pain, joint pain, fatigue, lethargy, weakness, dark skin (generally referred to as "bronze"), and loss of body hair. Administration of iRNA agents targeting TMPRSS6 can effectively treat one or more of these symptoms. Other symptoms associated with iron overload include an increased risk of liver disease (cirrhosis, liver cancer), heart attack or heart failure, diabetes, osteoarthritis, osteoporosis, metabolic syndrome, hypothyroidism, and hypogonadism, and in some cases, premature death. Iron mismanagement that causes iron overload can also accelerate neurodegenerative diseases such as Alzheimer's disease, early-onset Parkinson's disease, Huntington's disease, epilepsy, and multiple sclerosis. Administration of iRNAs targeting TMPRSS6, such as those listed in Tables 1 or 2, can treat one or more of these symptoms or prevent the development or progression of diseases or conditions that are exacerbated by increased iron levels. The method of this invention relates to the use of iRNA agents or pharmaceutical compositions thereof, such as for the treatment of conditions related to iron overload, in combination with other drugs and / or other treatments, such as those currently used to treat these conditions. For example, in some cases, iRNA agents targeting TMPRSS6 are administered in combination with iron chelators (e.g., deferoxamine), folic acid, blood transfusions, phlebotomy, agents for managing ulcers, agents for increasing fetal heme levels (e.g., hydroxyurea), agents for controlling infections (e.g., antibiotics and antiviral agents), agents for treating thrombotic states, or stem cell or bone marrow transplants. Stem cell transplants may use stem cells from related individuals such as siblings' umbilical cords. Examples of iron chelating agents include desferoxamine, exjade, deferiprone, vitamin E, wheat germ oil, water-soluble vitamin E (tocophersolan), and indicaxanthin. The iRNA agent and the additional therapeutic agent may be administered in the same composition via, for example, parenteral administration, or the additional therapeutic agent may be administered as part of a separate composition or by another method disclosed herein. Administration of the iRNA agent and the additional therapeutic agent may be performed simultaneously or at different times and in any order. Administration of the iRNA agent of this invention can reduce iron levels, reduce ferritin levels, and / or reduce transferrin saturation levels. For example, administration of the dsRNA can reduce serum iron levels and / or reduce serum ferritin levels. Transferrin saturation levels can be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. In another case, after administration, the reduction in transferrin saturation levels is maintained for 7, 10, 20, 30 days, or longer. Transferrin saturation levels can be reduced to below 50%, below 45%, below 40%, below 35%, below 35%, below 30%, below 25%, below 20%, below 15%, or lower. In another sample, this lower transferrin saturation level was maintained for 7, 10, 20, 30 days, or longer after administration. Transferrin saturation is a measure of the amount of iron bound to serum transferrin and corresponds to the ratio of serum iron to total iron binding capacity. Serum iron levels may be reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In another case, serum iron levels remained reduced for 7, 10, 20, 30 days, or longer after administration. Administration of the iRNA agent of the present invention preferably results in a decrease in blood iron levels, more specifically, serum iron levels, or iron levels in one or more tissues of a mammal. In some cases, iron levels decrease by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to pre-treatment levels. In this article, "reduction" means a statistically significant decrease in the level. This decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably a decrease to the level within the normal range for individuals without the condition. Administration of the iRNA agent of the present invention can increase serum hepcidin levels and / or increase hepcidin gene expression. For example, administration of the dsRNA can increase serum hepcidin by at least about 10%, 25%, 50%, 100%, 150%, 200%, 250%, 300%, or more. In yet another example, administration of the dsRNA can increase hepcidin mRNA levels by at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more. For example, the efficacy of treatment or prevention of disease can be assessed by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dosage of medicine required to maintain the therapeutic effect, the level of disease markers, or any other measurable parameter applicable to a specific disease to be treated or targeted for prevention. Monitoring the efficacy of treatment or prevention by measuring any one or any combination of these parameters is well within the capabilities of a person skilled in the art. For example, the efficacy of a particular treatment regimen can be evaluated by monitoring the level of transferrin saturation or serum transferrin levels. Iron level tests are typically performed on a patient's blood sample. Iron level tests measure the amount of iron in serum carried by the protein transferrin. The TIBC (Total Iron-Binding Capacity) test measures the amount of iron carried by the blood when transferrin is fully saturated. Since transferrin is produced by the liver, TIBC can be used to monitor liver function and nutrition. This transferrin test refers to the measurement of transferrin (also known as iron-transferring serum protein) levels in the blood. Transferrin saturation levels can be calculated by dividing serum iron levels by TIBC. This transferrin test measures the level of proteins in the blood used to store iron for the body's subsequent uses. The iRNA therapy described in this article can be used to treat individuals suffering from TMPRSS6-related conditions, such as those with elevated iron levels, as indicated by serum iron levels exceeding 350 μg / dL, 500 μg / dL, 1000 μg / dL, or higher. In a single-state sample, elevated serum iron levels are defined as exceeding 15, 20, 25, or 30 mg / g dry weight. The iRNA therapy described in this article can also be used to treat individuals with elevated iron levels, as indicated by elevated serum ferritin levels, such as measurements exceeding 300 μg / L, 500 μg / L, 1000 μg / L, 1500 μg / L, 2000 μg / L, 2500 μg / L, or 3000 μg / L or higher. The iRNA therapy described in this article can be further used to treat individuals with elevated iron levels, as indicated by elevated serum transferrin levels, such as measurements exceeding 400 mg / dL, 500 mg / L, 1000 mg / dL, or higher. The iRNA therapy described in this article can also be used to treat individuals with moderately elevated iron levels, as indicated by moderately elevated transferrin saturation levels such as 40%, 45%, or 50%, or higher. Furthermore, the therapy described in this article can also be used to prevent elevated iron levels in individuals with only a slight increase in transferrin saturation. Those skilled in this art can easily monitor transferrin saturation levels in subjects receiving the iRNA therapy described in this article and determine a decrease in transferrin saturation levels of at least 5% or 10%. The iRNA therapy described in this article can be used to treat individuals with elevated iron levels, as indicated by TIBC values ​​exceeding 400 μg / dL, 500 μg / dL, 1000 μg / dL, or higher. In certain states, the systems requiring treatment with the iRNA agent of the present invention have reduced hematocrit levels, reduced heme levels, increased red blood cell distribution width, increased reticulocyte count, reduced mature red blood cells, increased unsaturated iron binding, reduced inactive red blood cell production, reduced extramedullary hematopoiesis, and / or reduced HAMP1 expression levels. Patients can be further monitored using blood glucose (glucose) levels or alpha-fetoprotein levels, via ultrasound electrocardiography (e.g., to check cardiac function), electrocardiography (ECG) (e.g., to observe cardiac electrical activity), imaging tests (e.g., CT scans, MRI, and ultrasound), and liver function tests. Excess iron staining or iron concentration can be measured on liver biopsy samples or used to confirm the degree of liver damage, such as the stage of liver disease. A therapeutic or preventative effect is confirmed when there is a statistically significant improvement in one or more parameters of a disease state, or by the absence of worsening or development of unexpected symptoms. For example, a desirable change of at least 10%, and preferably 20%, 30%, 40%, 50%, or higher, in a measurable parameter of the disease can be an indicator of effective treatment. The efficacy of specific iRNA drugs of the present invention or formulations thereof can also be determined using experimental animal models of specific diseases known in the art. When using experimental animal models, therapeutic efficacy is confirmed when a statistically significant decrease in markers or symptoms is observed. Alternatively, this efficacy can be measured by a reduction in disease severity as determined by a person skilled in diagnostic techniques based on a clinically acceptable disease grading. In this article, "subject" includes humans or non-human animals, preferably vertebrates, and even more preferably mammals. Subjects may include genetically modified organisms. Ideally, the subject should be human, such as someone who has or is susceptible to TMPRSS6-related conditions. In certain embodiments of the method of the present invention, TMPRSS6 expression is reduced over an extended duration, such as at least 1 week, 2 weeks, 3 weeks, or 4 weeks or longer. For example, in some embodiments, by administering the iRNA agent disclosed herein, the expression of the TMPRSS6 gene is inhibited by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%. In some embodiments, by administering the iRNA agent, the TMPRSS6 gene is inhibited by at least about 60%, 70%, or 80%. In some embodiments, by administering the double-stranded oligonucleotide, the TMPRSS6 gene is inhibited by at least about 85%, 90%, or 95%. In another sample, the TMPRSS6 gene remained suppress...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a pharmaceutically acceptable salt thereof that inhibits the expression of the transmembrane protease serine 6 (TMPRSS6) in cells, comprising a positive and an antisense strand forming a double-stranded region, wherein, This antisense line contains at least 15 adjacent nucleotides, which are related to AAGGGCAGCUGAGCUCACCdTdT (SEQ ID NO). The nucleotide sequence of NO: 1381 differs by no more than 3 nucleotides, wherein all nucleotides of the positive and negative strands are modified nucleotides selected from the group consisting of 2'-O-methyl (2'-OMe) modified nucleotides, 2'-deoxy-2'-fluoro (2'-F) modified nucleotides and 2'-deoxy-modified nucleotides, wherein each strand is independently 15 to 30 nucleotides long, wherein the dsRNA agent contains 6 to 8 nucleotide links of thiophosphates, wherein the antisense strand contains two nucleotide links of thiophosphates at the 5'-terminus and two nucleotide links of thiophosphates at the 3'-terminus, and the positive strand contains at least two nucleotide links of thiophosphates at the 5'-terminus, and wherein the dsRNA agent is conjugated to an N-acetylglucosamine (GalNAc) derivative ligand at the 3'-terminus of the positive strand.

2. The dsRNA agent or a pharmaceutically acceptable salt thereof as described in claim 1, wherein, Each strand consists of 17 to 25 nucleotides independently.

3. The dsRNA agent or a pharmaceutically acceptable salt thereof as described in claim 1, wherein, At least one strand contains a dangling end of at least one nucleotide.

4. The dsRNA agent or a pharmaceutically acceptable salt thereof as described in claim 1, wherein, This ligand system consists of one or more GalNAc derivatives linked by divalent and trivalent branched linkages.

5. The dsRNA agent or a pharmaceutically acceptable salt thereof as described in claim 4, wherein, This formulation 6. The dsRNA agent as described in claim 5 or a pharmaceutically acceptable salt thereof, wherein, The dsRNA system is shown in the figure below, which binds to the ligand, and in which the X-line is O or S.

7. The dsRNA agent or a pharmaceutically acceptable salt thereof as described in claim 6, wherein, X series O.

8. The dsRNA agent as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein, The positive nucleotide sequence contains at least 13 adjacent nucleotides, and its nucleotide sequence differs from that of GGUGAGCUCAGCUGCCCUUdTdT (SEQ ID NO: 1047) by no more than 3 nucleotides.

9. The dsRNA agent as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein, The antisense string contains at least 12 adjacent nucleotides of the nucleotide sequence AAGGGCAGCUGAGCUCACCdTdT (SEQ ID NO: 1381).

10. The dsRNA agent as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein, The positive nucleotide sequence comprises at least 13 adjacent nucleotides that differ from the nucleotide sequence of GGUGAGCUCAGCUGCCCUUdTdT (SEQ ID NO: 1047) by no more than 3 nucleotides, and wherein the antisense nucleotide sequence comprises 12 adjacent nucleotides of the nucleotide sequence AAGGGCAGCUGAGCUCACCdTdT (SEQ ID NO: 1381).

11. The dsRNA agent as described in claim 10 or a pharmaceutically acceptable salt thereof, wherein, The positive nucleotide sequence comprises 10 adjacent nucleotides of the nucleotide sequence GGUGAGCUCAGCUGCCCUUdTdT (SEQ ID NO: 1047), and the negative nucleotide sequence comprises 12 adjacent nucleotides of the nucleotide sequence AAGGGCAGCUGAGCUCACCdTdT (SEQ ID NO: 1381).

12. A pharmaceutical composition for inhibiting the expression of a gene encoding the transmembrane protease serine 6 (TMPRSS6), comprising a dsRNA agent as described in any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof.

13. The pharmaceutical composition as described in claim 12, wherein, The dsRNA agent or its pharmaceutically acceptable salt is contained in a non-buffered solution.

14. The pharmaceutical composition as described in claim 13, wherein, The non-buffered solution is either salt water or water.

15. The pharmaceutical composition as described in claim 12, wherein, The dsRNA agent or its pharmaceutically acceptable salt is contained in a buffer solution.

16. The pharmaceutical composition as described in claim 15, wherein, The buffer solution contains acetate, citrate, prolyl protein, carbonate, or phosphate, or any combination thereof.

17. The pharmaceutical composition as described in claim 16, wherein, The buffer solution is phosphate-buffered saline (PBS).

18. An isolated cell containing a dsRNA agent or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 11.

19. A method for inhibiting the expression of TMPRSS6 in cells in vitro, the method comprising: (a) exposing cells in vitro to a dsRNA agent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as described in any one of claims 1 to 11; and (b) maintaining the cells prepared in step (a) in vitro for a time sufficient to allow degradation of the mRNA transcript of the TMPRSS6 gene, thereby inhibiting the expression of the TMPRSS6 gene in the cells.

20. The method as described in claim 19, wherein, The TMPRSS6 expression was suppressed by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%; serum hepcidin concentration was increased by at least 10%, 25%, 50%, 100%, 150%, 200%, 250%, or 300%; serum iron concentration was decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%; or transferrin saturation percentage was decreased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%.

21. Use of a therapeutically effective amount of any of the dsRNA agents described in claims 1 to 11 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in any of claims 12 to 17, in the preparation of a medicament for treating a subject suffering from TMPRSS6-related conditions.

22. The use as described in claim 21, wherein, The subject of the test is a person.

23. The use as described in claim 21 or 22, wherein, The TMPRSS6-related conditions include hereditary hemochromatosis, β-thalassemia, erythropoietinosis, or conditions related to iron overload.

24. The use as described in claim 23, wherein, This β-thalassemia lineage is severe thalassemia.

25. The use as described in claim 23, wherein, This β-thalassemia lineage is intermediate thalassemia.

26. The use as described in claim 23, wherein, The conditions associated with iron overload are Parkinson's disease, Alzheimer's disease, or Friedrich's ataxia.

27. The use as described in claim 21 or 22, which is for preparing a medicament suitable for treating subjects suffering from TMPRSS6-related conditions, wherein, This drug is suitable for subcutaneous administration.

28. The use as described in claim 21 or 22, which is for preparing a medicament suitable for treating subjects suffering from TMPRSS6-related conditions, wherein, This drug is suitable for intravenous administration.

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