Compositions and methods for inhibiting the expression of tmprss6 genes

Abstract

To provide compositions and methods for inhibiting TMPRSS6 gene expression.SOLUTION: Double-stranded ribonucleic acids (dsRNA) for inhibiting TMPRSS6 expression comprise a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarily with TMPRSS6 transcripts comprising at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense sequences listed in Table 2, 3 or 4 herein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS The present application of U.S. Provisional Patent Application No. 61 / 468,830, filed March 29, 2011, and U.S. Provisional Patent Application No. 61 / 568,942, filed December 9, 2011. The contents of these prior applications are incorporated herein by reference in their entirety.

[0002] The present invention relates to the specific inhibition of expression of the TMPRSS6 gene. [Background technology]

[0003] TMPRSS6 (transmembrane protease, serine 6) encodes a type II serine protease that is primarily expressed in the liver. TMPRSS6 affects iron levels in the liver by binding to and proteolytically cleaving the hepcidin activator and BMP co-receptor HJV (hemojuvelin), causing downregulation of hepcidin levels.

[0004] TMPRSS6 consists of a short N-terminal cytoplasmic tail, a type II transmembrane domain, a stem region composed of two extracellular CUB (complement factors C1s / C1r, sea urchin embryonic growth factor, and BMP (bone morphogenetic protein)) domains, three LDLR (low-density lipoprotein receptor class A) domains, and a C-terminal trypsin-like serine protease domain. It also contains consensus sites for N-glycosylation in the extracellular domain and potential phosphorylation sites in the cytoplasmic tail. [Brief explanation of the drawings]

[0005] [Figure 1] This is the sequence of human TMPRSS6 mRNA (reference sequence NM_153609.2, GI:56682967, recorded on January 23, 2011, SEQ ID NO: 1). [Figure 2A]1 depicts the efficacy of two chemically modified TMPRSS6-targeting siRNAs in reducing TMPRSS6 gene expression in mouse primary hepatocytes. [Figure 2B] 1 depicts the efficacy of two chemically modified TMPRSS6-targeting siRNAs in reducing TMPRSS6 gene expression in mouse primary hepatocytes. [Figure 3A] 1 depicts the effects of LNP-TMPRSS6 siRNA-1 (AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286) on TMPRSS6 and HAMP1 gene expression in WT C57BL / 6 mice, respectively. [Figure 3B] 1 depicts the effects of LNP-TMPRSS6 siRNA-1 (AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286) on TMPRSS6 and HAMP1 gene expression in WT C57BL / 6 mice, respectively. [Figure 4] 1 depicts the duration of TMPRSS6 siRNA-mediated effects on TMPRSS6 gene expression, HAMP1 gene expression, and serum iron levels in WT C57BL / 6 mice. [Figure 5] Depicts the level of TMPRSS6 siRNA-mediated TMPRSS6 silencing required to maintain TMPRSS6 siRNA-mediated effects on HAMP1 gene expression and serum iron levels in WT C57BL / 6 mice. [Figure 6A] Figure 6A depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hemoglobin (HBG) in WT C57BL / 6 mice at 6 hours, 24 hours, 48 ​​hours, 72 hours, 7 days, and 14 days after administration. [Figure 6B]Figure 6B depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hematocrit in WT C57BL / 6 mice 6 hours, 24 hours, 48 ​​hours, 72 hours, 7 days, and 14 days after administration. [Figure 7] Depict the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on serum iron parameters, including serum iron levels, unsaturated iron binding capacity (UIBC) levels, and transferrin saturation levels, in thalassemia mice (Th3 / +). [Figure 8A] 8A depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on reticulocyte and erythrocyte parameters in thalassemia mice (Th3 / +). FIG. 8A depicts the effect (%) on reticulocyte counts. [Figure 8B] Figure 8B depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on reticulocyte and erythrocyte parameters in thalassemia mice (Th3 / +). Figure 8B depicts the effect on hemoglobin content of reticulocytes (CHr). [Figure 8C] Figure 8A depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on reticulocyte and erythrocyte parameters in thalassemia mice (Th3 / +). Figure 8B depicts the effect on mature erythrocyte counts. [Figure 9A] 9A depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hematological parameters in thalassemia mice (Th3 / +). FIG. 9A depicts the effect on hematocrit (HCT) levels. [Figure 9B]Figure 9B depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hematological parameters in thalassemia mice (Th3 / +). Figure 9B depicts the effect on hemoglobin (HGB). [Figure 9C] Figure 9C depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hematological parameters in thalassemia mice (Th3 / +). Figure 9C depicts the effect on red blood cell (RBC) distribution width (RDW). [Figure 9D] Figure 9D depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hematological parameters in thalassemia mice (Th3 / +). Figure 9C depicts the effect on mean corpuscle volume (MCV). [Figure 10A] 10A depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on spleen and liver iron content in thalassemia mice (Th3 / +). FIG. 10B depicts the effect on total spleen iron content. [Figure 10B] Figure 10B depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on spleen and liver iron content in thalassemia mice (Th3 / +). Figure 10B depicts the effect on spleen weight. [Figure 10C] Figure 10C depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on spleen and liver iron content in thalassemia mice (Th3 / +). Figure 10C depicts the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on iron concentration in the liver. DETAILED DESCRIPTION OF THE INVENTION

[0006] Described herein are compositions and methods for causing RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the TMPRSS6 gene in cells, mammals, etc. Also described are compositions and methods for treating pathological conditions and diseases caused by the expression of the TMPRSS6 gene, such as disorders characterized by iron overload (e.g., thalassemias such as beta thalassemia intermedia or alpha thalassemia). Also described are compositions and methods for reducing or preventing iron absorption or mobilization, thereby improving iron overload in certain pathological conditions. The methods and compositions described herein are generally useful for treating hemochromatosis (body iron accumulation).

[0007] As used herein, the term "iRNA" refers to an agent that contains RNA as defined herein and mediates the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. In one embodiment, the iRNA described herein inhibits the expression of TMPRSS6 in cells or mammals. Alternatively, in another embodiment, the iRNA upregulates the expression of TMPRSS6 in cells or mammals.

[0008] The iRNAs included in the compositions described herein include double-stranded ribonucleic acid (dsRNA) having an RNA strand (antisense strand) with a region of 30 nucleotides or less, typically 19-24 nucleotides in length, that is substantially complementary to at least a portion of an mRNA transcript of the TMPRSS6 gene. In one embodiment, the dsRNA comprises a region of at least 15 contiguous nucleotides.

[0009] In one embodiment, an iRNA that inhibits expression of the TMPRSS6 gene comprises at least two complementary sequences. The iRNA comprises a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand comprises a nucleotide sequence substantially complementary to at least a portion of an mRNA encoding TMPRSS6, with the region of complementarity being 30 nucleotides or less and at least 15 nucleotides in length. Typically, the iRNA is 19-24 nucleotides in length, e.g., 19-21 nucleotides in length. In some embodiments, the iRNA is about 15 to about 25 nucleotides in length, and in other embodiments, the iRNA is about 25 to about 30 nucleotides in length. When assayed, such as by the methods described herein, the iRNA inhibits TMPRSS6 gene expression by at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% or more upon contact with a cell expressing TMPRSS6. In one embodiment, the TMPRSS6 iRNA is formulated into a stable nucleic acid lipid particle (SNALP).

[0010] In one embodiment, an iRNA provided herein comprises a first sequence of a dsRNA selected from the group consisting of the sense sequences of Table 2, 3, or 4, and a second sequence selected from the group consisting of the antisense sequences of Table 2, 3, or 4. The iRNA molecules provided herein may comprise natural nucleotides or may contain at least one modified nucleotide, including, but not limited to, 2'-O-methyl modified nucleotides, nucleotides having a 5'-phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and unnatural base-containing nucleotides. Generally, such modified sequences are based on a first sequence of the iRNA selected from the group consisting of the sense sequences of Table 2, 3 or 4, and a second sequence selected from the group consisting of the antisense sequences of Table 2, 3 or 4.

[0011] In one embodiment, an iRNA featured herein comprises a sense strand of a TMPRSS6 dsRNA having a sequence selected from the group consisting of SEQ ID NO:111, SEQ ID NO:455, SEQ ID NO:109, SEQ ID NO:524, SEQ ID NO:89, SEQ ID NO:494, SEQ ID NO:445, SEQ ID NO:592, SEQ ID NO:47, and SEQ ID NO:540; and an antisense strand consisting of a sequence selected from the group consisting of SEQ ID NO:112, SEQ ID NO:456, SEQ ID NO:110, SEQ ID NO:525, SEQ ID NO:90, SEQ ID NO:495, SEQ ID NO:446, SEQ ID NO:593, SEQ ID NO:48, and SEQ ID NO:541.

[0012] In another embodiment, the composition that contains TMPRSS6 targeting dsRNA is administered to the subject with elevated iron level, for example, elevated liver iron level.The subject with elevated iron level can be identified as the subject with elevated serum iron level (for example, above 350 μ g / dL, above 500 μ g / dL, or above 1000 μ g / dL), elevated serum ferritin level, or the subject with transferrin saturation level above 40%, above 45%, above 50%, or above 60%.

[0013] Mild to moderate iron overload is suggested by serum ferritin levels between 300 and 2500 μg / L, while levels above 2500 μg / L are associated with an increased risk of heart disease. Serum ferritin levels above 1000 μg / L are associated with adverse outcomes in both primary and secondary iron overload. Serum ferritin levels above 200 μg / L in premenopausal women and above 300 μg / L in men and postmenopausal women suggest primary iron overload due to hemochromatosis, and ferritin levels above 1000 μg / L typically suggest liver damage due to iron overload. Subjects with serum ferritin levels greater than 300 μg / L, 500 μg / L, 1000 μg / L, 1500 μg / L, 2000 μg / L, or 2500 μg / L are candidates for treatment with TMPRSS6-targeting dsRNA.

[0014] In another embodiment, a subject with elevated transferrin levels (e.g., transferrin levels greater than 400 mg / dL, greater than 500 mg / dL, greater than 1000 mg / dL) is administered a composition containing a TMPRSS6-targeting dsRNA.

[0015] Iron level can also be measured by TIBC (Total Iron Binding Capacity) test. TIBC test measures the amount of iron that blood carries when transferrin is fully saturated. Because transferrin is produced by the liver, TIBC can be used to monitor liver function and nutrition. Subjects with TIBC values ​​greater than 400 μg / dL, greater than 500 μg / dL, or greater than 1000 μg / dL are candidates for treatment with TMPRSS6-targeted dsRNA.

[0016] In one embodiment, administration of dsRNA reduces the iron level in liver or serum by at least 5%, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more.In some embodiments, compared with the level before treatment, one or more of serum ferritin level, serum transferrin level, transferrin saturation level or TIBC value is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more.In another embodiment, the reduction in iron level, serum ferritin level, transferrin or transferrin saturation level or TIBC value is maintained for at least 5, 10, 20, 30, or 40 days or longer.

[0017] In one embodiment, subjects are selected, at least in part, based on their need for iron level reduction (as opposed to simply selecting patients who happen to need it). In one embodiment, the iRNA described herein targets a wild-type TMPRSS6 RNA transcript, while in another embodiment, the iRNA targets a mutant transcript (e.g., a TMPRSS6 RNA harboring an allelic variant). For example, the iRNA featured herein can target a polymorphic variant, such as a single nucleotide polymorphism (SNP), of TMPRSS6. In another embodiment, the iRNA targets both wild-type and mutant TMPRSS6 transcripts. In yet another embodiment, the iRNA targets a transcript variant of TMPRSS6.

[0018] In one embodiment, an iRNA featured in the present invention targets a non-coding region of the TMPRSS6 RNA transcript, such as the 5' or 3' untranslated region. In one embodiment, an iRNA featured in the invention is delivered to the liver, e.g., to hepatocytes of the liver, or to Kupffer cells, e.g., hypertrophic Kupffer cells.

[0019] In one aspect, embodiments featured herein provide a cell containing at least one iRNA featured herein. The cell is generally a mammalian cell, such as a human cell. In another aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of the TMPRSS6 gene in an organism, typically a human subject. The composition typically comprises one or more of the iRNAs described herein and a pharmaceutically acceptable carrier or delivery vehicle. In one embodiment, the composition is used to treat disorders that cause increased iron levels, such as hemochromatosis. For example, the composition is useful for treating thalassemia, such as beta-thalassemia intermedia.

[0020] In another embodiment, the pharmaceutical composition is formulated for administration of a dosing regimen described herein, e.g., not more than once every two months, not more than once a month, not more than twice a month, not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once a week, etc. In another embodiment, administration of the pharmaceutical composition can continue for more than one month, e.g., for 1, 2, 3, or 6 months, or 1 year, or 5 years, or 10 years, or longer, for the remainder of the subject's lifespan.

[0021] In another embodiment, a composition containing an iRNA described herein, such as a TMPRSS6-targeting dsRNA, is administered in conjunction with a non-iRNA therapeutic agent, such as an agent known to treat hemochromatosis or a disorder that causes hemochromatosis, such as thalassemia. For example, an iRNA featured in the invention can be administered in conjunction with an agent for treating beta thalassemia, such as beta thalassemia intermedia, or another disorder associated with elevated iron levels.

[0022] In another embodiment, a TMPRSS6 iRNA is administered to a patient, followed by a non-iRNA agent (or vice versa). In another embodiment, a TMPRSS6 iRNA and a non-iRNA therapeutic agent are administered simultaneously. In one embodiment, the agent is an agent that affects iron levels, such as, for example, an iron chelator (e.g., desferrioxamine) or folic acid.

[0023] In another aspect, provided herein is a method for inhibiting expression of the TMPRSS6 gene in a cell by performing the following steps: (a) introducing into a cell double-stranded ribonucleic acid (dsRNA) containing at least two sequences complementary to each other, the dsRNA including a sense strand having a first sequence and an antisense strand having a second sequence, the antisense strand having a complementary region substantially complementary to at least a portion of an mRNA encoding TMPRSS6, the complementary region being 30 nucleotides or less, i.e., 15 to 30 nucleotides in length, generally 19 to 24 nucleotides in length, and the dsRNA inhibits expression of the TMPRSS6 gene by at least 10%, preferably at least 20%, at least 30%, or at least 40% or more upon contact with a cell expressing TMPRSS6; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the TMPRSS6 gene, thereby inhibiting expression of the TMPRSS6 gene in the cells.

[0024] In another aspect, the present invention provides methods and compositions useful for activating expression of the TMPRSS6 gene in a cell or mammal. In another aspect, the present disclosure provides a method for regulating expression of the TMPRSS6 gene in a cell by performing the following steps: (a) introducing into a cell double-stranded ribonucleic acid (dsRNA) comprising at least two sequences complementary to each other, the dsRNA comprising a sense strand having a first sequence and an antisense strand having a second sequence, the antisense strand having a complementary region substantially complementary to at least a portion of an mRNA encoding TMPRSS6, the complementary region being 30 nucleotides or less, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, and the dsRNA modulates TMPRSS6 gene expression by at least 10%, preferably at least 20%, at least 30%, or at least 40% or more upon contact with a cell expressing TMPRSS6; (b) maintaining the cells produced in step (a) for a time sufficient to allow degradation and protection of the mRNA transcripts of the TMPRSS6 gene, thereby regulating expression of the TMPRSS6 gene in the cells.

[0025] In one embodiment, the method is for inhibiting gene expression in liver cells, such as hepatocytes or Kupffer cells, hi another embodiment, the method is for activating gene expression in liver cells.

[0026] In another aspect, the present invention provides methods for treating, preventing, ameliorating, or managing pathological processes mediated by TMPRSS6 expression, such as hemochromatosis-related disorders. In one embodiment, the method comprises administering a therapeutically or prophylactically effective amount of one or more iRNAs featured in the present invention to a patient in need of such treatment, prevention, amelioration, or management. In one embodiment, the patient has a thalassemia, such as beta-thalassemia intermedia. In another embodiment, administration of the TMPRSS6-targeting iRNA alleviates or reduces the severity of at least one symptom of the TMPRSS6-mediated disorder in the patient, e.g., an iron-overload-related symptom such as joint pain, abdominal pain, or weakness.

[0027] In one aspect, the present invention provides a vector for inhibiting expression of the TMPRSS6 gene in a cell. In one embodiment, the vector comprises at least one regulatory sequence operably linked to a nucleotide sequence encoding at least one strand of an iRNA described herein.

[0028] In another aspect, the present invention provides a cell containing a vector for inhibiting expression of the TMPRSS6 gene in a cell, the vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding at least one strand of one of the iRNAs described herein.

[0029] In yet another aspect, the present invention provides a composition useful for treating diseases such as β-thalassemia, comprising a TMPRSS6 iRNA combined with a second iRNA targeting a second gene involved in the pathological condition. For example, the second iRNA may target a hypoxia-inducible factor, such as HIF-1a or HIF-2a; GDF15; or a negative regulator of hepcidin, such as TWSG1. In one embodiment, the second iRNA targets a gene involved in a second disorder caused by β-thalassemia. For example, the second iRNA may target a gene involved in diabetes, thrombosis, or osteopenia.

[0030] Details of various embodiments of the invention are set forth in the description that follows. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0031] Described herein are iRNAs and methods for using them to inhibit the expression of the TMPRSS6 gene in cells or mammals, wherein the iRNA targets the TMPRSS6 gene. Also provided are compositions and methods for treating pathological conditions and diseases caused by TMPRSS6 gene expression, such as elevated iron-level-related conditions. The iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi: RNA interface). In alternative embodiments, the iRNA activates the expression of the TMPRSS6 gene in cells or mammals, wherein the iRNA targets the TMPRSS6 gene.

[0032] TMPRSS6 plays a key role in iron homeostasis as an inhibitor of HAMP gene expression. The HAMP gene encodes the hepatic hormone hepcidin, a central regulator of iron homeostasis. Hepcidin binds to the iron exporter protein ferroportin (FPN1), which is primarily localized in absorptive enterocytes, hepatocytes, and macrophages. Hepcidin binding to the extracellular domain of ferroportin leads to its internalization and degradation, thereby reducing dietary iron absorption from the intestine and iron release from macrophages and hepatocytes. HAMP gene expression can be stimulated in response to iron through a Bone Morphogenetic Protein (BMP) / Sons of Mothers Against Decapentaplegic (SMAD)-dependent signaling cascade mediated by the BMP coreceptor hemojuvelin (HJV). TMPRSS6's key role in HAMP regulation is to inhibit BMP-mediated HAMP upregulation. TMPRSS6 cleaves the BMP coreceptor HJV, which is essential for BMP-mediated HAMP upregulation; thus, it inhibits BMP-mediated HAMP upregulation by preventing BMP signaling, SMAD translocation to the nucleus, and HAMP transcriptional activation.

[0033] Several human and mouse studies have confirmed the role of TMPRSS6 in HAMP regulation and iron homeostasis (Du et al., Science 320:1088-1092, 2008; Folgueras et al., Blood 112:2539-45, 2008). Studies have shown that loss-of-function mutations in TMPRSS6 result in upregulation of hepcidin expression and can cause a hereditary iron deficiency anemia termed iron refractory iron deficiency anemia (IRIDA) (Finberg, Seminars in Hematology 2009, 46:378-86), which is characterized by elevated hepcidin levels, hypochromic microcytic anemia, low mean corpuscular volume (MCV), low transferrin saturation, poor oral iron absorption, and an incomplete response to parenteral iron. However, loss-of-function mutations in positive regulators of HAMP (e.g., BMP1, BMP4, and HFE) have been shown to downregulate hepcidin expression, leading to iron overload disorders (Milet et al., Am J Hum Gen 2007, 81:799-807; Finberg et al., Blood 2011, 117:4590-4599). In primary iron overload disorders, collectively referred to as hereditary hemochromatosis (HH), anemias characterized by massive ineffective hematopoiesis, and secondary hemochromatosis, such as β-thalassemia intermedia (TI), hepcidin levels are low despite elevated serum iron concentrations and iron stores.A mouse model of beta-thalassemia intermedia demonstrates that loss of TMPRSS6 expression results in elevated hepcidin levels (Finberg, 2010, Oral Presentation, "TMPRSS6, an inhibitor of hepatic BMP / Smad signaling, is required for hepcidin suppression and iron loading in a mouse model of beta-thalassemia," American Society of Hematology Annual Meeting, 2010, Abstract #164).

[0034] The present invention describes a method and iRNA composition for regulating TMPRSS6 gene expression.In certain embodiments, TMPRSS6-specific iRNA is used to reduce or inhibit TMPRSS6 expression, thereby leading to increased HAMP expression and reduced serum iron levels.Therefore, using the iRNA composition featured in the present invention to inhibit the expression or activity of TMPRSS6 gene can be a useful approach for therapeutic methods aimed at reducing iron levels in subjects.Such inhibition can be useful for treating disorders associated with elevated iron levels, such as hemochromatosis or thalassemia, for example, β-thalassemia.

[0035] The iRNAs of the compositions described herein comprise an RNA strand (antisense strand) that is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, and has a region that is substantially complementary to at least a portion of the mRNA transcript of the TMPRSS6 gene. The use of these iRNAs enables targeted degradation of the mRNA of genes implicated in pathologies associated with TMPRSS6 expression in mammals. In particular, very low doses of TMPRSS6 iRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of TMPRSS6 gene expression. Using cell-based assays, the inventors have demonstrated that TMPRSS6-targeting iRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of TMPRSS6 gene expression. Therefore, methods and compositions comprising these iRNAs are useful for treating pathological processes that can be mediated by downregulation of TMPRSS6, such as the treatment of disorders that cause elevated iron levels, such as hemochromatosis or β-thalassemia, e.g., β-thalassemia intermedia. The detailed description below discloses how to make and use iRNA-containing compositions to inhibit expression of the TMPRSS6 gene, as well as compositions and methods for treating diseases and disorders caused by expression of this gene.

[0036] Pharmaceutical composition embodiments featured herein also include iRNAs having an antisense strand that is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and that comprises a region that is substantially complementary to at least a portion of an RNA transcript of the TMPRSS6 gene, in combination with a pharmaceutically acceptable carrier. Composition embodiments featured herein also include iRNAs having an antisense strand that is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and that has a complementary region that is substantially complementary to at least a portion of an RNA transcript of the TMPRSS6 gene.

[0037] Thus, in some aspects, the present invention features a pharmaceutical composition containing TMPRSS6 iRNA and a pharmaceutically acceptable carrier, a method for inhibiting expression of the TMPRSS6 gene using the composition, and a method for treating a disease caused by expression of the TMPRSS6 gene using the pharmaceutical composition.

[0038] I. Definition For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. In the event of an apparent conflict between usage in other parts of this specification and its definition provided in this section, the definition in this section shall control.

[0039] "G", "C", "A", "T" and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases, respectively.However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or alternative replacement moieties, as will be further detailed below.Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially changing the base pairing properties of the oligonucleotides comprising nucleotides with such replacement moieties.As a non-limiting example, a nucleotide comprising inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil.Therefore, a nucleotide containing uracil, guanine or adenine can be replaced with a nucleotide containing inosine, for example, in the nucleotide sequence of the dsRNA described herein. In another example, adenine and cytosine can be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form a GU wobble base pair with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods described herein.

[0040] As used herein, "Transmembrane Protease, Serine 6" (TMPSSR6) refers to a specific polypeptide expressed in cells. TMPRSS6 is also known as matriptase-2, IRIDA (iron refractory iron-deficiency anemia), transmembrane protease serine 6, type II transmembrane serine protease 6, and membrane-bound mosaic serine proteinase matriptase-2. TMPRSS6 is a serine protease type II transmembrane protein approximately 899 amino acids in length. TMPRSS6 contains multiple domains, including a short internal region, a transmembrane domain, a sea urchin sperm protein / enteropeptidase domain / agrin (SEA) domain, two complement factor / urchin embryonic growth factor / BMP domains (CUB), three LDL-R class a domains (LDLa), and a trypsin-like serine protease domain with a conserved His-Asp-Ser triad (HDS). The sequence of the human TMPRSS6 mRNA transcript is NM_153609.2 (SEQ ID NO: 1) (Figure 1).

[0041] As used herein, the term "iRNA" refers to an agent that contains RNA, as defined herein, and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. In one embodiment, the iRNA described herein results in inhibition of TMPRSS6 expression. Alternatively, in another embodiment, the iRNA described herein activates TMPRSS6 expression.

[0042] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the TMPRSS6 gene, including messenger RNA (mRNA), which is the product of RNA processing of a primary transcript. A target portion of a sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, target sequences are generally 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all subranges therebetween. By way of non-limiting example, target sequences can be 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 ... The amino acid sequence may be 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

[0043] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0044] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions to form a double-stranded structure, as would be understood by one of skill in the art. Such conditions may be stringent conditions, which may include, for example, 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may be applied. One of skill in the art can determine the optimal set of conditions for testing the complementarity of two sequences depending on the end use of the hybridized nucleotides.

[0045] Complementary sequences in iRNAs, such as those in dsRNAs described herein, include base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs upon hybridization of a duplex of up to 30 base pairs (bp), while retaining the ability to hybridize under conditions most appropriate for their ultimate use, such as inhibiting gene expression through the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, if a dsRNA comprises one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, and the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, it may still be referred to as "fully complementary" for purposes described herein.

[0046] "Complementary" sequences, as used herein, may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, such as, but not limited to, G:U wobble base pairs or Hoogsteen base pairs, so long as the above requirements regarding their hybridization ability are met.

[0047] The terms "complementary," "fully complementary," and "substantially complementary" may be used herein to refer to matching bases between the sense and antisense strands of a dsRNA or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

[0048] As used herein, a polynucleotide "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of a target mRNA (e.g., an mRNA encoding TMPRSS6). For example, a polynucleotide is complementary to at least a portion of a TMPRSS6 mRNA if its sequence is substantially complementary to a non-interrupted portion of the mRNA encoding TMPRSS6.

[0049] The term "double-stranded RNA" or "dsRNA," as used herein, refers to an iRNA, including an RNA molecule or molecular complex, having a hybridized double-stranded region comprising two antiparallel and substantially complementary nucleic acid strands, said to have "sense" and "antisense" orientations with respect to the target RNA. The double-stranded region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, but typically ranges in length from 9 to 36 base pairs, e.g., 15 to 30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length within this range, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36, as well as 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-30 base pairs, 18-40 base pairs, 18-42 base pairs, 18-43 base pairs, 18-44 base pairs, 18-45 base pairs, 18-46 base pairs, 18-47 base pairs, 18-48 base pairs, 18-50 base pairs, 18-51 base pairs, 18-52 base pairs, 18-53 base pairs, 18-54 base pairs, 18-55 base pairs, 18-56 base pairs, 18-57 base pairs, 18-58 base pairs, 18-59 base pairs, 19-60 base pairs, 19-61 base pairs, 19-62 base pairs, 19-63 base pairs, 19-64 base pairs, 19-65 base pairs, 20-66 base pairs, 20-67 base pairs, 20-68 base pairs, 20-69 base pairs The length may be any subrange therebetween, including, but not limited to, 8-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNA generated in cells by processing with Dicer and similar enzymes is generally in the 19-22 base pair range. One strand of the double-stranded region of the dsDNA comprises a sequence that is substantially complementary to a region of the target RNA. The two strands that form the duplex structure can be derived from a single RNA molecule with at least one self-complementary region, or can be generated from two or more separate RNA molecules.When the double-stranded region is generated from two strands of a single molecule, the molecule can have a double-stranded region separated by a single strand of nucleotides (referred to herein as "hairpin loop") between the 3'-end of one strand that forms the double-stranded structure and the 5'-end of each other strand.The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments, the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.When the two substantially complementary strands of dsRNA are formed by another RNA molecule, these molecules can be, but do not necessarily have to be, covalently linked.When the two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a "linker".The term "siRNA" is also used herein to refer to the above-mentioned dsRNA.

[0050] Those skilled in the art will recognize that the terms "RNA molecule" or "ribonucleic acid molecule" encompass not only naturally expressed or found RNA molecules, but also RNA analogs and derivatives comprising one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, a "ribonucleoside" comprises a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside with one, two, or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleotide" can be considered equivalent as used herein. RNA can be modified in the nucleobase structure or in the ribose phosphate backbone structure, for example, as described herein below. However, molecules comprising ribonucleoside analogs or derivatives must retain the ability to form double strands. As non-limiting examples, the RNA molecule may also comprise at least one modified ribonucleoside, including, but not limited to, a 2'-O-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate or non-natural base-containing nucleoside, or any combination thereof. Alternatively, the RNA molecule may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more modified ribonucleosides up to the entire length of the dsRNA molecule. The modification may not be the same for each of such multiple modified ribonucleosides in an RNA molecule. In one embodiment, the modified RNA contemplated for use in the methods and compositions described herein is a peptide nucleic acid (PNA), which has the ability to form the required double-stranded structure to enable or mediate the specific degradation of a target RNA through the RISC pathway.

[0051] In one embodiment, modified ribonucleosides include deoxyribonucleosides.In such cases, iRNA agents can comprise one or more deoxynucleosides, including, for example, deoxynucleoside overhangs, or one or more deoxynucleosides within the double-stranded portion of dsRNA.However, it is clear that under all circumstances, double-stranded DNA molecules are not included in the term "iRNA".

[0052] In one embodiment, the RNA interference agent comprises a single-stranded RNA that interacts with a target RNA sequence and induces cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into plant and invertebrate cells is degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al., Genes Dev., 2001, 15:485). Dicer, a RNase III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein et al., 2001, Nature, 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen et al., 2001, Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir et al., 2001, Genes Dev. 15:188). Thus, in one aspect, the present invention relates to single-stranded RNAs that promote RISC complex formation, resulting in target gene silencing.

[0053] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. A nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide can be present on the 5'-end, the 3'-end, or both ends of either the antisense or sense strand of a dsRNA.

[0054] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end and / or 5'-end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3'-end and / or 5'-end. In another embodiment, one or more nucleotides in the overhang are substituted with a thiophosphate nucleoside.

[0055] The term "blunt-ended" or "blunt-ended" used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analogue at a given end of dsRNA, i.e., there is no nucleotide overhang.One or both ends of dsRNA can be blunt-ended.When both ends of dsRNA are blunt-ended, the dsRNA is said to be blunt-ended.For clarity, "blunt-ended" dsRNA is a dsRNA with both ends blunted, i.e., there is no nucleotide overhang at either end of the molecule.In most cases, such molecules are double-stranded throughout their entire length.

[0056] The term "antisense strand" or "guide strand" refers to an iRNA strand, such as a dsRNA, that includes a region that is substantially complementary to a target sequence. As used herein, the term "region complementary" refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence as defined herein. If the complementary region is not completely complementary to the target sequence, mismatches may exist in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, such as within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end.

[0057] The terms "sense strand" or "passenger strand," as used herein, refer to an iRNA strand that includes a region that is substantially complementary to a region of the antisense strand, as defined herein.

[0058] As used herein, in one embodiment, the term "SNALP" refers to a stable nucleic acid-lipid particle. SNALP refers to a lipid vesicle with a reducing aqueous interior containing a nucleic acid, such as an iRNA, or a plasmid from which the iRNA is transcribed. SNALPs are described, for example, in U.S. Patent Application Publication No. 20060240093, U.S. Patent Application Publication No. 20070135372, and International Publication No. 2009082817. Examples of "SNALP" formulations are described elsewhere herein.

[0059] "Introducing into a cell," when referring to iRNA, means facilitating or causing uptake or absorption into the cell, as understood by those skilled in the art. Absorption or uptake of iRNA can occur through unassisted diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; iRNA can also be "introduced into a cell" when the cell is part of a living organism. In such cases, introduction into a cell includes delivery to the organism, e.g., in vivo. For in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a β-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Patent Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below or known in the art.

[0060] As used herein, the term "modulate expression of" refers to at least partial "inhibition" or partial "activation" of TMPRSS6 gene expression in cells treated with an iRNA composition described herein compared to TMPRSS6 expression in untreated cells.

[0061] The terms "activate," "enhance," "upregulate expression," "increase expression," and the like, when referring to the TMPRSS6 gene, refer herein to at least partial activation of TMPRSS6 gene expression manifested by an increase in the amount of TMPRSS6 mRNA that can be isolated from or detected in a first cell or group of cells that have been treated to increase expression of the TMPRSS6 gene and in which the TMPRSS6 gene is transcribed, compared to a second cell or group of cells (control cells) that are substantially identical to the first cell or group of cells but have not been treated to increase expression of the TMPRSS6 gene.

[0062] In one embodiment, TMPRSS6 gene expression is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA described herein. In some embodiments, TMPRSS6 gene expression is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured herein. In some embodiments, TMPRSS6 gene expression is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA described herein. In some embodiments, TMPRSS6 gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold or more in cells treated with an iRNA described herein compared to expression in untreated cells. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006, Proc. Natl. Acad. Sci. USA, 103, 17337-42, and U.S. Patent Nos. 20070111963 and 2005226848, each of which is incorporated herein by reference.

[0063] The terms "silence," "inhibit expression," "downregulate expression," "suppress expression," and the like, when referring to the TMPRSS6 gene, refer herein to at least partial suppression of TMPRSS6 gene expression, as manifested by a reduction in the amount of TMPRSS6 mRNA isolated from or detectable in a first cell or group of cells that have been treated to inhibit expression of the TMPRSS6 gene and in which the TMPRSS6 gene is transcribed, compared to a second cell or group of cells (control cells) that are substantially identical to the first cell or group of cells but have not been treated to inhibit expression of the TMPRSS6 gene. The degree of inhibition typically refers to

[0064]

number

[0065] Alternatively, the degree of inhibition may be expressed in terms of a reduction in a parameter functionally linked to TMPRSS6 gene expression, such as the amount of protein encoded by the TMPRSS6 gene, or the number of cells displaying a certain phenotype, such as reduced iron levels or iron absorption. In principle, TMPRSS6 gene silencing can be determined in any cell that expresses TMPRSS6, either constitutively or by genome engineering, and by any suitable assay.

[0066] For example, in some cases, expression of the TMPRSS6 gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA featured herein. In some embodiments, the TMPRSS6 gene is suppressed by at least about 60%, 70%, or 80% by administration of an iRNA described herein. In some embodiments, the TMPRSS6 gene is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA described herein.

[0067] As used herein, in the context of TMPRSS6 expression, the terms "treat," "treatment," and the like refer to the alleviation or alleviation of a pathological process mediated by TMPRSS6 expression. In the context of the present invention, insofar as any of the other conditions listed herein below (other than a pathological process mediated by TMPRSS6 expression) are concerned, the terms "treat," "treatment," and the like refer to the alleviation or alleviation of at least one symptom associated with such condition, or the slowing or reversal of the progression or expected progression of such condition, such as the slowing of the progression of hemochromatosis, such as thalassemia.

[0068] By "reduce," in the context of a disease marker or symptom, is meant a statistically significant decrease in such level, which can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, preferably to a level considered to be within the normal range for an individual with such disorder.

[0069] As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a pathological process mediated by TMPRSS6 expression or overt symptoms of a pathological process mediated by TMPRSS6 expression. The specific therapeutically effective amount can be readily determined by an ordinary practitioner and can vary depending on factors known in the art, such as the type of pathological process mediated by TMPRSS6 expression, the patient's medical history and age, the stage of the pathological process mediated by TMPRSS6 expression, and the administration of other agents that inhibit the pathological process mediated by TMPRSS6 expression.

[0070] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to an amount of an iRNA that is effective to produce the intended pharmacological, therapeutic, or preventative result. For example, if a given clinical treatment is considered effective if there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, then a therapeutically effective amount of a drug for treating that disease or disorder is the amount necessary to produce at least a 10% reduction in the parameter. For example, a therapeutically effective amount of a TMPRSS6-targeting iRNA may reduce TMPRSS6 protein levels by at least 10%.

[0071] As used herein, the term "thalassemia" refers to a recessively inherited blood disorder. Loss-of-function mutations result in reduced or no synthesis of one of the globin chains that make up hemoglobin, resulting in a deficiency of normal globin protein. Patients with thalassemia have a deficiency of either alpha globin (referred to as alpha thalassemia), beta globin (referred to as beta thalassemia), or, rarely, delta globin. In alpha thalassemia, excess beta chains form unstable tetramers with abnormal oxygen dissociation curves. Beta thalassemia can be mild, severe, or intermediate.

[0072] The beta-globin chain is encoded by a single gene called the HBB (hemoglobin, beta) gene. Beta-thalassemia minor occurs in patients who carry one mutant beta-thalassemia allele and one wild-type allele. This condition does not affect blood iron levels, and patients do not require treatment. Beta-thalassemia major results when patients carry two knockout mutant beta-thalassemia alleles. These patients accumulate excess iron, which is stored primarily in hypertrophic Kupffer cells. Patients with beta-thalassemia major are typically treated with chronic transfusion therapy, iron chelation, splenectomy, and allogeneic hematopoietic cell transplantation. Beta-thalassemia intermedia results when patients carry one knockout allele and one partial loss-of-function allele of the beta-thalassemia gene. These patients accumulate excess iron, which is stored primarily in liver parenchymal cells. Patients with thalassemia major and thalassemia intermedia have anemia (hypoxia), which leads to increased EPO (erythropoietin) and thus dramatic compensatory ineffective hematopoiesis (production of red blood cells by stem cells in the bone marrow). Patients with thalassemia intermedia sometimes develop hepatosplenomegaly, jaundice, osteopenia, thrombotic events, lower extremity ulcers, pulmonary hypotension, congestive heart failure, diabetes, growth hormone deficiency, hypothyroidism, hypoparathyroidism, hypogonadism, and facial dysmorphism.

[0073] As used herein, the term "hemochromatosis" refers to a disorder that results in excessive iron absorption from the gastrointestinal tract. Hemochromatosis occurs in two forms: primary and secondary. Primary hemochromatosis, the most common genetic disorder in the United States (affecting approximately 1 in 200-300 Americans), is usually caused by a specific genetic problem that results in excessive iron absorption. Secondary, or acquired, hemochromatosis can be caused by diseases such as thalassemia or sideroblastic anemia. Secondary hemochromatosis sometimes occurs in patients with hemolytic anemia and chronic alcoholism. Symptoms of hemochromatosis include abdominal pain, joint pain, fatigue, lack of energy, weakness, darkening of the skin (often referred to as "bronzing"), and hair loss.

[0074] The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, inert diluents such as pharmaceutically acceptable excipients, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while cornstarch and alginic acid are suitable disintegrants. Binders include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The active ingredients contained in the formulation are described in more detail herein below.

[0075] As used herein, a "subject" is a mammal, such as, for example, dogs, horses, cats, and other non-human primates. In a preferred embodiment, the subject is a human. As used herein, the term "LNPXX" (where "XX" is a number) is also referred to herein as "AFXX." For example, LNP09 is also referred to as AF09, and LNP12 is also known as and referred to as AF12.

[0076] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components essential to the invention, but still open to the inclusion of unspecified elements, whether essential or not.

[0077] As used herein, the term "consisting essentially of" refers to elements essential to a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment featured in this invention.

[0078] The term "consisting of" refers to the compositions, methods, and their respective components described herein, and is exclusive of any element not recited in that description of an embodiment.

[0079] II. Double-stranded ribonucleic acid (dsRNA) Described herein are iRNA agents that regulate expression of the TMPRSS6 gene. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the TMPRSS6 gene in a cell or mammal, such as a human with elevated iron levels, e.g., a patient with β-thalassemia or hemachromatosis. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of an mRNA formed during expression of the TMPRSS6 gene. The complementary region is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and the dsRNA, upon contact with a cell expressing the TMPRSS6 gene, inhibits expression of the TMPRSS6 gene by at least 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or protein-based methods such as Western blot. In one embodiment, the iRNA agent activates expression of the TMPRSS6 gene in the cell or mammal. Expression of the TMPRSS6 gene in cell cultures such as COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cells, or in biological samples from subjects can be assayed by measuring TMPRSS6 mRNA levels, such as by bDNA or TaqMan® assays, or by measuring protein levels, such as by immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0080] dsRNA contains two RNA strands that are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary to the target sequence, generally perfectly complementary. The target sequence can be derived from the sequence of mRNA formed during expression of the TMPRSS6 gene. The other strand (the sense strand) contains a region complementary to the antisense strand such that, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. Typically, the double-stranded structure is 15-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. Similarly, the region of complementarity to the target sequence is 15-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 nucleotides in length. In some embodiments, the dsRNA is 15-20 nucleotides in length, and in other embodiments, the dsRNA is 25-30 nucleotides in length. As those skilled in the art will recognize, the target region of an RNA targeted for cleavage is most often a portion of a larger RNA molecule, which is often an mRNA molecule. Where applicable, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNAs with duplexes as short as 9 base pairs can, in some circumstances, mediate RNAi-directed RNA cleavage. In most cases, the target is at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0081] Those skilled in the art will also recognize that the double-stranded region is the primary functional portion of a dsRNA, e.g., a 9-36 base pair double-stranded region, e.g., 15-30 base pairs. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs is a dsRNA, as long as it is processed into a functional duplex, e.g., 15-30 base pairs, that targets a desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting TMPRSS6 expression is not generated in a target cell by cleavage of a larger dsRNA.

[0082] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs.The dsRNA can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer, such as that commercially available from Biosearch (Applied Biosystems, Inc.), as will be further discussed below.In one embodiment, the TMPRSS6 gene is the human TMPRSS6 gene.In another embodiment, the TMPRSS6 gene is the mouse or rat TMPRSS6 gene.The sequence of mouse TMPRSS6 mRNA is in GenBank accession number NM_027902 (GI:125656151, archived December 28, 2010).The sequence of rat TMPRSS6 mRNA is in GenBank accession number NM_001130556.1 (GI:194474097, archived January 17, 2011). In certain embodiments, the first sequence is the sense strand of a dsRNA comprising a sense sequence of Table 2, 3, or 4, and the second sequence is the antisense strand of a dsRNA comprising an antisense sequence of Table 2, 3, or 4. Alternative dsRNA agents targeting other locations within the target sequences provided in Table 2, 3, or 4 can be readily determined using the target sequences and the flanking TMPRSS6 sequences.

[0083] In one embodiment, the dsRNA comprises at least two nucleotide sequences, a sense and an antisense sequence, wherein the sense strand is selected from the group of sequences provided in Tables 2, 3, or 4. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to an mRNA sequence produced during expression of the TMPRSS6 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide designated in Tables 2, 3, or 4 as the sense strand and the second oligonucleotide designated as the antisense strand that corresponds to the sense strand from Tables 2, 3, or 4. As described elsewhere herein and as known in the art, the complementary sequences of the dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0084] Those skilled in the art are well aware that dsRNAs having a duplex structure of 20-23 base pairs, particularly 21 base pairs, have been advocated as being particularly effective in inducing RNA interference (Elbashir et al., European Molecular Biology Organization (EMBO), 2001, Vol. 20, pp. 6877-6888). However, others have found that shorter or longer RNA duplex structures can be similarly effective. In the above-described embodiments, due to the nature of the oligonucleotide sequences provided in Tables 2, 3, or 4, the dsRNAs described herein can contain at least one strand of at least 21 nt in length. It can be reasonably expected that shorter duplexes having one of the sequences in Tables 2, 3, or 4, with only a few nucleotides missing from one or both termini, may be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a subsequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one of the sequences of Tables 2, 3 or 4, and whose ability to inhibit expression of the TMPRSS6 gene differs by no more than 5, 10, 15, 20, 25, or 30% from inhibition from a dsRNA comprising the full-length sequence, are contemplated according to the present invention.

[0085] In addition, the RNAs provided in Tables 2, 3, or 4 identify sites in the TMPRSS6 transcript that are susceptible to RISC-mediated cleavage. Accordingly, the present invention further features iRNAs that target within one of these sequences. As used herein, an iRNA is said to target a specific site in an RNA transcript if it promotes cleavage of the transcript anywhere within that site. Such iRNAs generally contain at least 15 consecutive nucleotides from one of the sequences provided in Tables 2, 3, or 4, linked to additional nucleotide sequences incorporated from regions adjacent to the selected sequence in the TMPRSS6 gene.

[0086] Target sequences are generally 15-30 nucleotides in length, although there is wide variability in the suitability of specific sequences within this range to induce cleavage of any given target RNA. While the various software packages and guidelines presented herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed, either physically or figuratively (e.g., by computer simulation), around the target RNA sequence to identify sequences within a size range that may serve as target sequences. By successively shifting the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, while the sequences identified in, for example, Tables 2, 3 or 4 represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by successively "window walking" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

[0087] It is contemplated that further optimization of any sequence identified, for example, in Tables 2, 3, or 4, can be achieved by systematically adding or removing nucleotides to create longer or shorter sequences, and then testing these created sequences by walking through windows of a size longer or shorter than the target RNA from that position. Again, combining this approach of creating new target candidates with testing the effectiveness of iRNAs based on these target sequences in inhibition assays known in the art or described herein can lead to further improvements in inhibition efficiency. Still further, such optimized sequences can be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or modifying overhangs, or other modifications known in the art and / or discussed herein to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermostability, enhancing transmembrane delivery, targeting specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0088] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the range of mismatches is not located in the center of the complementary region. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches be limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23-nucleotide iRNA agent RNA strand complementary to a TMPRSS6 gene region, the RNA strand generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an iRNA containing mismatches with the target sequence is effective in inhibiting TMPRSS6 gene expression. Examining the effectiveness of iRNAs with mismatches in inhibiting TMPRSS6 gene expression is important, especially when a specific complementary region of the TMPRSS6 gene is known to have polymorphic sequence variation within the population.

[0089] In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4 nucleotides, generally 1 or 2 nucleotides. Such dsRNAs with at least one nucleotide overhang have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial properties. Nucleic acids featured in the present invention can be synthesized using methods described in "Current protocols in nucleic acid chemistry," which are incorporated herein by reference. The RNA compounds may be synthesized and / or modified by methods established in the art, such as those described in "Theoretical Chemistry," edited by Beaucage, S.L. et al., John Wiley & Sons, Inc., New York, NY, USA. Modifications include, for example, (a) terminal modifications, such as 5'-end modifications (phosphorylation, conjugated linkage, inverted linkage) and 3'-end modifications (conjugated linkage, DNA nucleotide, inverted linkage, etc.); (b) base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base that base pairs with an expanded repertoire of partners, base removal (abasic nucleotide), or conjugated base; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and (d) backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or RNAs that do not contain natural internucleoside linkages. The RNA with modified backbone particularly includes that which does not have phosphorus atom in backbone.For the purpose of this specification, and as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in their internucleoside backbone is also considered to be oligonucleoside.In certain embodiments, the modified RNA has phosphorus atom in its internucleoside backbone.

[0090] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and boranophosphates with reversed polarity, in which adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0091] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include U.S. Pat. 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, each of which is incorporated herein by reference. ;U.S. Patent Nos. 5,321,131; U.S. Patent No. 5,399,676; U.S. Patent No. 5,405,939; U.S. Patent No. 5,453,496; U.S. Patent No. 5,455,233; U.S. Patent No. 5,466,677; U.S. Patent No. 5,476,925; U.S. Patent No. 5,519,126; U.S. Patent No. 5,536,821; U.S. Patent No. 5,541,316; U.S. Patent No. 5,550,111; U.S. Patent No. 5,563,253; U.S. Patent No. 5,571,79 No. 9; U.S. Patent No. 5,587,361; U.S. Patent No. 5,625,050; U.S. Patent No. 6,028,188; U.S. Patent No. 6,124,445; U.S. Patent No. 6,160,109; U.S. Patent No. 6,169,170; U.S. Patent No. 6,172,209; U.S. Patent No. 6,239,265; U.S. Patent No. 6,277,603; U.S. Patent No. 6,326,199; U.S. Patent No. 6,346,614; U.S. Patent No. 6,444,423; U.S. Patent No. 6,5 Nos. 31,590; 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 RE39464.

[0092] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, those with amide backbones, and others with mixed N, O, S, and CH2 components.

[0093] Representative United States patents that teach the preparation of the above oligonucleosides include U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967, each of which is incorporated herein by reference. ; U.S. Patent No. 5,489,677; U.S. Patent No. 5,541,307; U.S. Patent No. 5,561,225; U.S. Patent No. 5,596,086; U.S. Patent No. 5,602,240; U.S. Patent No. 5,608,046; U.S. Patent No. 5,610,289; U.S. Patent No. 5,618,704; U.S. Patent No. 5,623,070; U.S. Patent No. 5,663,312; U.S. Patent No. 5,633,360; U.S. Patent No. 5,677,437; and U.S. Patent No. 5,677,439.

[0094] In other RNA mimics suitable or contemplated for use in iRNA, both the sugar and internucleoside linkages, the backbone of the nucleotide units, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds is found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0095] Some embodiments featured herein include RNAs with phosphorothioate backbones, and oligonucleosides with heteroatom backbones that are, in particular, -CH-NH-CH-, -CH-N(CH)-O-CH- (known as methylene(methylimino) or MMI backbones), -CH-ON(CH)-CH-, -CH-N(CH)-N(CH)-CH-, and -N(CH)-CH-CH- (natural phosphodiester backbones are represented as -OPO-CH-) of the aforementioned U.S. Patent No. 5,489,677, and with amide backbones of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, RNAs featured herein have morpholino backbone structures of the aforementioned U.S. Patent No. 5,034,506.

[0096] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs, such as dsRNAs provided herein, can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl, or C2-C 10 It may be an alkenyl or alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In another embodiment, the dsRNA includes one of the following at the 2' position: C1 to C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intervening agent, group that improves the pharmacokinetic properties of iRNA, or group that improves the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta 78:486-504, 1995), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, or O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and the 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), or 2'-O-CH2-O-CH2-N(CH2)2, as also described herein below in the Examples.

[0097] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, specifically at the 3' position of the sugar on the 3'-terminal nucleotide, or in 2'-5'-linked dsRNA, and at the 5' position of the 5'-terminal nucleotide. An iRNA can also have a sugar mimic, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of the above modified sugar structures include, but are not limited to, U.S. Pat. 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; and 5,519,134, each of which is incorporated herein by reference and certain of which are commonly owned with the present application. Nos. 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920.

[0098] iRNAs may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil (pseudouracil); 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine (5-hydroxymethylcytosine); xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil (pseudouracil); 4-thiouracil (pseudouracil); 4-thiouracil (pseudouracil); 4-thiouracil (pseudouracil); 4- Other synthetic and natural nucleobases include uracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-daazaadenine; and 3-deazaguanine and 3-deazaadenine. Further examples of nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in "Modified Nucleosides in Biochemistry, Biotechnology and Medicine", edited by Herdewijn, P., Wiley-VCH, 2008; and those disclosed in "The Concise Encyclopedia of Polymer Science and Engineering". Concise Encyclopedia Of Polymer Science and those disclosed by Sanghvi, Y. S., Chapter 15, "dsRNA Research and Applications," pp. 289-302, Crooke, S. T. and Lebleu, B. (eds.), CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS; Crooke, ST; and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them exemplary base substitutions, even more so when combined with 2'-O-methoxyethyl sugar modifications.

[0099] Representative U.S. patents that teach the preparation of the above-mentioned specific modified nucleobases as well as other modified nucleobases include U.S. Pat. No. 3,687,808, as noted above, and the following U.S. Pat. Nos.: 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594 ,121, 5,596,091; U.S. Patent Nos. 5,614,617; 5,681,941; U.S. Patent No. 6,015,886; U.S. Patent No. 6,147,200; U.S. Patent No. 6,166,197; U.S. Patent No. 6,222,025; U.S. Patent No. 6,235,887; U.S. Patent No. 6,380,368; U.S. Patent No. 6,528,640; U.S. Patent No. 6,639,062; U.S. Patent No. 6,617,438; U.S. Patent No. 7,045,610; U.S. Patent No. 7,427,672; and U.S. Patent No. 7,495,088, and U.S. Patent No. 5,750,692, which is also incorporated herein by reference.

[0100] The RNA of an iRNA can also be modified to contain one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties, in which the ribose moiety comprises an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce nonspecific effects (Elmen, J. et al., 2005, Nucleic Acids Research, Vol. 33, No. 1, pp. 439-447; Mook, O.R. et al., 2007, Mol Cancer Ther., Vol. 6, No. 3, pp. 833-843; Grunweller, A. et al., 2003, Nucleic Acids Research, Vol. 1, pp. 439-447). Acids Research, Vol. 31, No. 12, pp. 3185-3193).

[0101] Representative United States patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the contents of each of which are incorporated herein by reference in their entirety.

[0102] Another modification of the RNA of the iRNA featured in the present invention involves chemically linking to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA.Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proceedings of the National Academy of Sciences, 1989, Vol. 86, pp. 6553-6556); cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, Vol. 4, pp. 1053-1060); e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, Vol. 660, pp. 306-309; Manoharan et al., Bioorganic Medical Chemistry, 1994, Vol. 1060-1060); thioethers such as thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538); dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Letters, 1991); Lett., 1990, 259, pp. 327-330; Svinarchuk et al., Biochimie, 1993, 75, pp. 49-54); aliphatic chains such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, pp. 3651-3654; Shea et al., Nucl. Acids Research, 1996, 259, pp. 327-330). Res., 1990, Vol. 18, pp. 3777-3783); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides). Nucleotides, 1995, 14, pp. 969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, pp. 3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, pp. 229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, pp. 923-937).

[0103] In one embodiment, the ligand alters the distribution, targeting, or lifespan of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand provides improved affinity for a selected target, such as a molecule, a cell, or cell type (e.g., a liver cell, such as a hepatocyte), a compartment, such as a subcellular or organ compartment, a tissue, organ, or region of the body, for example, compared to a species in the absence of such a ligand. Preferred ligands do not participate in double-strand pairing in duplexed nucleic acids.

[0104] Ligands can include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines are polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or alpha helical peptides.

[0105] The ligand can also include a targeting group such as a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody that binds to a specific cell type such as a kidney cell. The targeting group can be thyroid stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetylglucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidomimetic.

[0106] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithophosphate, and the like. Examples of suitable cleavage inhibitors include acetylcholinesterases (e.g., acetylcholinesterase ...

[0107] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for co-ligands; 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 lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0108] The ligand can be a substance, such as a drug, that can increase uptake of an iRNA agent into a cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments, e.g., by disrupting the cell's cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0109] In some embodiments, the ligand attached to the iRNA described herein functions as a PK modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides comprising several phosphorothioate linkages are also known to bind to serum proteins; therefore, short oligonucleotides, such as, for example, about 5-, 10-, 15-, or 20-base oligonucleotides comprising multiple phosphorothioate linkages in their backbones, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. In addition, aptamers that bind to serum components (eg, serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0110] For macromolecular and hydrophilic drug molecules that cannot easily cross bilayer membranes, entrapment within the endosomal / lysosomal compartments of cells is considered the greatest hurdle to their effective delivery to their site of action. In recent years, several approaches and strategies have been devised to address this issue. In liposomal formulations, the most common approach is the use of fusogenic lipids in the formulation (Singh, RS, Goncalves, C. et al., 2004, "On the Gene Delivery Efficacies of pH-Sensitive Cationic Lipids via Endosomal Protonation. A Chemical Biology Investigation," Chem. Biol., Vol. 11, pp. 713-723). Other components that exhibit pH-sensitive endosomolytic activity through protonation and / or pH-induced conformational changes include charged polymers and peptides. See, for example, Hoffman, AS, Stayton, PS et al., 2002, "Design of 'smart' polymers that can direct intracellular drug delivery." 13, pp. 992-999; Kakudo, Chaki T., S. et al., 2004, "Transferrin-Modified Liposomes Equipped with a pH-Sensitive Fusogenic Peptide: An Artificial Viral-like Delivery System," Biochemistry, 436, pp. 5618-5628; Yessine, M.A. and Leroux, J.C., 2004, "Membrane-destabilizing polyanions: interaction with lipid bilayers and endosomal escape of biomacromolecules," Advanced Adv. Drug Deliv. Rev., Vol. 56, pp. 999-1021; Oliveira, S., van Rooy, I. et al., 2007, "Fusogenic peptides enhance endosomal escape improving iRNA-induced silencing of oncogenes," Int. J. Pharm., Vol. 331, pp. 211-214. They are commonly used in the context of drug delivery systems such as liposomes or lipoplexes. For example, in folate receptor-mediated delivery using liposomal formulations, pH-sensitive fusogenic peptides incorporated into liposomes have been shown to enhance activity through improved drug release during the uptake process (Turk, MJ, Reddy, JA et al., 2002).Biochim. Biophys. Acta, Vol. 1559, pp. 56-68, describes the characterization of a novel pH-sensitive peptide that promotes drug release from liposomes that target folic acid at endosomal pH.

[0111] In certain embodiments, the endosomolytic component of the present invention can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and / or fusogenicity. Peptidomimetics can be small protein-like chains designed to mimic peptides. Peptidomimetics can result from the modification of existing peptides to alter the molecular properties or the synthesis of peptide-like molecules using unnatural amino acids or their analogs. In certain embodiments, they have improved stability and / or biological activity compared to peptides. In certain embodiments, the endosomolytic component adopts its active conformation at endosomal pH (e.g., pH 5-6). An "active" conformation is one in which the endosomolytic component promotes lysis of endosomes and / or promotes transport of the modular composition featured herein or any of its components (e.g., nucleic acids) from the endosome to the cytoplasm.

[0112] Hemolysis assays can be used to screen compound libraries for differential membrane activity at endosomal pH versus neutral pH. Promising candidates isolated by this method can be used as components of the modular compositions featured in the present invention. A method for identifying endosomolytic components for use in the compositions and methods of the present invention can include providing a compound library; contacting blood cells with library members, wherein the pH of the medium in which the contacting occurs is controlled; and determining whether the compounds induce differential lysis of blood cells at low pH (e.g., about pH 5-6) versus neutral pH (e.g., about pH 7-8).

[0113] Exemplary endosomolytic components include GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972), EALA peptide (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 certain embodiments, the endosomolytic component may contain a chemical group (e.g., an amino acid) that changes charge or protonation in response to a pH change. The endosomolytic component may be linear or branched. Exemplary primary sequences of endosomolytic components include H2N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO2H (SEQ ID NO: 2); H2N-(AALAEALAEALAEALAEALAEALAAAAGGC)-CO2H (SEQ ID NO: 3); and H2N-(ALEALAEALEALAEA)-CONH2 (SEQ ID NO: 4).

[0114] In certain embodiments, two or more endosomolytic components can be incorporated into the iRNA agents featured in the present invention. In some embodiments, this involves incorporating two or more identical endosomolytic components into the iRNA agent. In other embodiments, this involves incorporating two or more different endosomolytic components into the iRNA agent.

[0115] These endosomolytic components can mediate endosomal leakage, for example, by changing their conformation at endosomal pH. In certain embodiments, endosomolytic components exist in a random coil conformation at neutral pH and can rearrange into an amphipathic helix at endosomal pH. This conformational transition may result in these peptides inserting into the lipid membrane of endosomes, causing leakage of endosomal contents into the cytoplasm. Because the conformational transition is pH-dependent, endosomolytic components exhibit little fusogenic activity while circulating in the blood (pH approximately 7.4). "Fusogenic activity," as used herein, is defined as the activity of an endosomolytic component that results in lipid membrane disruption. One example of fusogenic activity is the disruption of the endosomal membrane by an endosomolytic component, resulting in endosomal lysis or leakage and the transport of one or more components (e.g., nucleic acids) of the modular compositions featured herein from the endosome to the cytoplasm.

[0116] In addition to the hemolytic assay described herein, those skilled in the art can use other methods to test and identify suitable endosomolytic components. For example, the ability of a compound to respond to changes in charge, etc., depending on the pH environment can be tested by conventional methods, for example, in a cell assay. In certain embodiments, a test compound is combined or contacted with cells so that the cells internalize the test compound, for example, by endocytosis. An endosome preparation can then be made from the contacted cells, and the endosome preparation can be compared with an endosome preparation from control cells. A change, such as a decrease in the endosomal fraction of the contacted cells compared to the control cells, suggests that the test compound can function as a fusogen. Alternatively, the contacted cells and the control cells can be evaluated by microscopy, for example, light or electron microscopy, to determine differences in the endosomal population in the cells. The test compound and / or endosomes can be labeled to, for example, quantify endosomal leakage.

[0117] In another type of assay, one or more test or putative fusogenic factors are used to construct the iRNA agents described herein. The iRNA agents can be labeled for easy visualization. Once the iRNA agent is taken up into the cell, the ability of endosomolytic components to promote endosomal leakage can be assessed, for example, by preparing an endosomal preparation or by microscopy techniques to visualize the labeled iRNA agent in the cytoplasm. In certain other embodiments, inhibition of gene expression or any other physiological parameter can be used as a surrogate marker for endosomal leakage.

[0118] In another embodiment, circular dichroism spectroscopy can be used to identify compounds that exhibit pH-dependent structural transitions. A two-tiered assay may also be performed, where a first assay evaluates the ability of a test compound alone to respond to a pH change, and a second assay evaluates the ability of a modular composition comprising the test compound to respond to a pH change.

[0119] lipid complex In one ligand, the ligand or complex is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of complex to target tissue, for example, non-renal target tissue of the body.For example, the target tissue can be the liver, including the parenchymal cells of the liver.Other molecules that can bind to HSA can also be used as ligand.For example, neproxin or aspirin can be used.Lipid or lipid-based ligand can (a) increase the degradation resistance of complex, (b) increase the targeting or transport to target cell or cell membrane, and / or (c) can be used to regulate the binding of serum protein, for example, HSA.

[0120] For example, lipid-based ligand can be used for regulation, such as controlling the binding of complex to target tissue.For example, the lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to kidney, and therefore less likely to be removed from body.The lipid or lipid-based ligand that binds weaker to HSA can be used to target complex to kidney.

[0121] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed.

[0122] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells may also be used in place of or in addition to the lipid-based ligand.

[0123] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant types, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0124] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the cell-penetrating agent is amphipathic. An exemplary cell-penetrating agent is a peptide such as tat or antennopedia. When the cell-penetrating agent is a peptide, it can be modified, including peptidylmimetic, invertomer, non-peptide or pseudo-peptide bond, and D-amino acid use. The helical agent is preferably an α-helical agent with a lipophilic and lipophobic phase.

[0125] Cell-penetrating peptides Peptides suitable for use in the present invention can be natural peptides, such as tat or antennapedia peptides, synthetic peptides, or peptidomimetics. Furthermore, peptides can be modified peptides, e.g., peptides can comprise non-peptide or pseudopeptide bonds and D-amino acids. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. The addition of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by facilitating cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0126] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 5). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 6)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting numerous polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 7)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 8)) have been shown to function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as peptides identified from a phage-display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). Preferably, the lipid-tethered peptide or peptidomimetic is a cell-targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or an RGD mimetic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications to increase stability or induce conformational properties. Any of the structural modifications described below can be utilized.

[0127] RGD peptide moieties 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). RGD peptides can facilitate targeting of dsRNA agents to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues. For example, glycosylated RGD peptides can deliver iRNA agents to tumor cells expressing αvβ3 (Haubner et al., Jour. Nucl. Med. 42:326-336, 2001).

[0128] Peptides that target markers abundant in proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly cells that display αββ integrin. Thus, RGD peptides, RGD-containing cyclic peptides, RGD peptides containing D-amino acids, and synthetic RGD mimetics can be used. In addition to RGD, other moieties that target integrin ligands can be used. In general, such ligands can be used to control proliferating cells and angiogenesis.

[0129] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two key amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain nuclear localization signals (NLSs). For example, cell-penetrating peptides can be bisected amphipathic peptides, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res., 31, 2717-2724, 2003).

[0130] Carbohydrate complex In some embodiments, the iRNA oligonucleotides described herein further comprise a carbohydrate complex. Carbohydrate complexes are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, "carbohydrate" refers to either a carbohydrate itself, composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having a carbohydrate moiety composed of one or more monosaccharide units (which may be linear, branched, or cyclic), each having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include C5 and higher (preferably C5-C8) sugars; disaccharides and trisaccharides, including sugars having two or three monosaccharide units (preferably C5-C8).

[0131] In one embodiment, the carbohydrate conjugate is selected from the group consisting of the following: Formula II-Formula XXII:

[0132] [ka]

[0133] [ka]

[0134] [ka]

[0135] [ka]

[0136] [ka] Other exemplary carbohydrate complexes for use in the embodiments described herein include:

[0137] [ka] (wherein one of X or Y is an oligonucleotide, and the other is hydrogen), but is not limited thereto.

[0138] In some embodiments, the carbohydrate conjugate further comprises other ligands, such as, but not limited to, PK modulators, endosomolytic ligands, and cell-penetrating peptides.

[0139] Linker In some embodiments, the conjugates described herein may be attached to the iRNA oligonucleotide by a variety of linkers, which may be cleavable or non-cleavable.

[0140] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound. A linker is typically a direct bond or an atom such as oxygen or sulfur, NR 8, C(O), C(O)NH, SO, SO2, SO2NH, or units such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhetero ...alkynyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl, alkylheteroarylalkynyl, and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R 8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (wherein R 8is hydrogen, acyl, aliphatic, or substituted aliphatic). In one embodiment, the linker is 1 to 24 atoms, preferably 4 to 24 atoms, preferably 6 to 18 atoms, more preferably 8 to 18 atoms, and most preferably 8 to 16 atoms.

[0141] A cleavable tether is one that is sufficiently stable extracellularly but is cleaved upon entry into a target cell to release the two moieties tethered by the linker. In preferred embodiments, the cleavable tether is cleaved at least 10 times more rapidly, and preferably at least 100 times more rapidly, in the target cell, or under first standard conditions (e.g., which may be selected to mimic or correspond to intracellular conditions), than in the subject's blood, or under second standard conditions (e.g., which may be selected to mimic or correspond to conditions found in blood or serum).

[0142] Cleavable linking groups are susceptible to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common in cells than in serum or blood, or are found at higher levels or activity. Examples of such degradable agents include oxidizing or reducing enzymes or reducing agents such as mercaptans present in cells, which can degrade redox-cleavable linking groups by reduction, and are selective for specific substrates or do not have substrate specificity; esterases; agents that can create an acidic environment, such as endosomes or those that produce a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate specific), and phosphatases.

[0143] Cleavable linking groups, such as disulfide bonds, can be highly sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand in the cell or to a desired compartment of the cell.

[0144] Linker can contain cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can depend on the cell to be targeted.For example, the ligand for targeting liver can be linked to cationic lipid through a linker that contains ester group.Hepatocytes are rich in esterase, therefore linker is more efficiently cleaved in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0145] Linkers containing peptide bonds may be used in targeting peptidase-rich cell types such as hepatocytes and synoviocytes. In general, the suitability of candidate cleavable linkers can be evaluated by testing the ability of degradable agents (conditions) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, with the first condition selected to indicate cleavage in target cells and the second condition selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10, or 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0146] Redox-cleavable linking groups One class of cleavable linkers is redox-cleavable linkers that are cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can rely on the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium compared to conditions selected to mimic the extracellular medium.

[0147] Phosphate-Based Cleavable Tethers Phosphate-based cleavable linkers can be cleaved by an agent that decomposes or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group in a cell is an enzyme such as a phosphatase in the cell. Examples of phosphate-based linkers are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0148] Acid-cleavable linking group An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment of about pH 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or by an agent such as an enzyme that can act as a general acid. In cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups have the general formula -C=NN- 、It may have -C(O)O, or -OC(O). Preferred embodiments are aryl groups, where the carbon is attached to the oxygen of the ester (alkoxy groups); substituted alkyl groups; or tertiary alkyl groups such as dimethylpentyl or t-butyl. These candidates may be evaluated using methods similar to those described above.

[0149] Ester-based linking groups Ester-based cleavable linkers are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0150] Peptide-based cleaving groups Peptide-based cleavable linkers are cleaved in cells by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula -NHCHR A C(O)NHCHR B C(O)-(wherein, R A and R B are the R groups of two adjacent amino acids). These candidates can be evaluated using methods similar to those described above.

[0151] Representative carbohydrate conjugates that can be attached to a linker include:

[0152] [ka]

[0153] [ka]

[0154] [ka] (wherein one of X or Y is an oligonucleotide, and the other is hydrogen), but is not limited thereto.

[0155] Representative United States patents that teach the preparation of RNA complexes include U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; and 5,109,124, each of which is incorporated herein by reference. Details; U.S. Patent Nos. 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941 Nos.; U.S. Patent Nos. 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506 ;U.S. Patent No. 5,262,536;U.S. Patent No. 5,272,250;U.S. Patent No. 5,292,873;U.S. Patent No. 5,317,098;U.S. Patent Nos. 5,371,241, 5,391,723;U.S. Patent Nos. 5,416,203, 5,451,463;U.S. Patent No. 5,510,475;U.S. Patent No. 5,512,667;U.S. Patent No. 5,514,785;U.S. Patent No. 5,565,552;U.S. Patent No. 5,567,810;U.S. Patent No. 5,574,142;Examples of patents that may be used include, but are not limited to, U.S. Patent Nos. 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; and 7,037,646.

[0156] Not all positions in a given compound need be uniformly modified; in fact, two or more of the aforementioned modifications may be incorporated in a single compound, or even in a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. "Chimeric" iRNA compounds or "chimeras," in the context of the present invention, are iRNA compounds, preferably dsRNA, that contain two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of dsRNA compounds. These iRNAs typically contain at least one region in which the RNA is modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As one example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, shorter iRNAs often produce comparable results compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

[0157] In some cases, the RNA of an iRNA may be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNA to enhance iRNA activity, cellular distribution, or intracellular uptake; procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, Vol. 365, No. 1, pp. 54-61; Letsinger et al., Proceedings of the National Academy of Sciences, 1989, Vol. 86, p. 6553), cholic acid (Manoharan et al., Bioorganic Medical Chemistry, 2007, Vol. 86, p. 6553), and thiamin monophosphate (Thiamin Monophosphate). Bioorg. Med. Chem. Let., 1994, 4, 1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Research, 1994, 4, 1053), Res., 1992, 20, 533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 1001).49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 14, 969). Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing amino linkers at one or more positions in the sequence. The amino groups are then reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction may be carried out in solution phase while the RNA is still bound to the solid support, or following RNA cleavage. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0158] iRNA delivery Delivery of iRNA to a subject in need thereof can be achieved in several different ways. In vivo delivery can be achieved directly by administering a composition comprising an iRNA, such as dsRNA, to a subject. Alternatively, delivery can be achieved indirectly by administering one or more vectors that encode and induce expression of the iRNA.

[0159] Direct Delivery of iRNA Compositions Generally, any method for delivering nucleic acid molecules can be adapted for use with iRNA (see, e.g., Akhtar S. and Julian RL., 1992, Trends Cell. Biol., Vol. 2, No. 5, pp. 139-144, and International Publication No. WO 94 / 02595, the entire contents of which are incorporated herein by reference). However, there are three important factors to consider for successful in vivo delivery of iRNA molecules: (a) the biological stability of the delivered molecule, (2) prevention of nonspecific effects, and (3) accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or implantation into tissues (such as, but not limited to, tumors), or by administering the preparation locally. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure of systemic tissues that might otherwise be harmed by or degrade the agent, and allows for the administration of lower total doses of iRNA molecules. Several studies have demonstrated successful gene product knockdown when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ, et al., 2004, Retina 24, pp. 132-138) and by subretinal injection in mice (Reich, SJ, et al., 2003, Mol. Vis. 9, pp. 210-216) both prevented neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice could reduce tumor volume (Pille, J. et al., 2005, Mol. Ther., 11, pp. 267-274) and extend the survival time of tumor-bearing mice (Kim, WJ. et al., 2006, Mol. Ther., 14, pp. 343-350; Li, S. et al., 2007, Mol. Ther., 15, pp. 515-523).RNA interference can be delivered to the CNS by direct injection (Dorn, G. et al., 2004, Nucleic Acids 32:e49; Tan, P.H. et al., 2005, Gene Ther. 12:59-66; Makimura, H. et al., 2002, BMC Neurosci. 3:18; Shishkina, G.T. et al., 2004, Neuroscience 129:521-528; Thakker, E.R. et al., 2004, Proceedings of the National Academy of Sciences 101:17270-17275; Akaneya, Y. et al., 2005, Journal of Neuroscience 101:17270-17275). Successful local delivery has been demonstrated to the lungs via intranasal administration (Howard, K. A. et al., 2006, Mol. Ther., 14, 476-484; Zhang, X. et al., 2004, J. Biol. Chem., 279, 10677-10684; Bitko, V. et al., 2005, Nat. Med., 11, 50-55). To treat disease, iRNA can be administered systemically, and RNA can be modified, or alternatively, delivered using a drug delivery system; both methods prevent rapid degradation of dsRNA by endogenous endo- and exonucleases.Modification of RNA or pharmaceutical carriers can also enable iRNA compositions to be targeted to target tissues, avoiding undesirable non-specific effects.iRNA molecules can be modified by chemically binding lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation.For example, systemic injection of iRNAs directed against ApoB conjugated to lipophilic cholesterol moieties into mice resulted in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J. et al., 2004, Nature 432, pp. 173-178). Conjugation of iRNAs to aptamers has been shown to suppress tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J.O. et al., 2006, Nature Biotechnol. 24, pp. 1005-1015). In alternative embodiments, iRNAs can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of iRNA molecules (which are negatively charged) and also enhance their interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be conjugated to iRNA or induced to form vesicles or micelles that encase iRNA (see, for example, Kim SH. et al., 2008, Journal of Controlled Release, Vol. 129, No. 2, pp. 107-116). The formation of vesicles or micelles further prevents degradation of iRNA upon systemic administration. Methods for making and administering cationic iRNA complexes are well within the capabilities of one of ordinary skill in the art (see, e.g., Sorensen, D.R. et al., 2003, J. Mol. Biol. 327:761-766; Verma, U.N. et al., 2003, Clin. Cancer Res. 9:1291-1300; Arnold, A.S. et al., 2007, J. Hypertens. 25:197-205, the contents of which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R. et al., 2003, supra; Verma, U.N. et al., 2003, supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S. et al., 2006, Nature 441, pp. 111-114), cardiolipin (Chien, P.Y. et al., 2005, Cancer Gene Ther. 12, pp. 321-328; Pal, A. et al., 2005, International Journal of Oncology 10, pp. 111-114), and ribozymes (Chien, P.Y. et al., 2005, Cancer Gene Ther. 12, pp. 321-328). J. Oncol. 26, pp. 1087-1091), polyethyleneimine (Bonnet ME et al., 2008, Pharmaceutical Research (Pharm. Res.), August 16, advance online publication; Aigner A., ​​2006, J. Biomed. Biotechnol., pp. 71659), Arg-Gly-Asp (RGD) peptide (Liu S., 2006, Mol. Pharm. 3, pp. 472-487), and polyamidoamine (Tomalia DA et al., 2007, Biochemical Society Biochem. Soc. Trans. 35, pp. 61-67; Yoo, H. et al., 1999, Pharm. Res. 16, pp. 1799-1804. In some embodiments, for systemic administration, iRNA is complexed with cyclodextrin. Methods and pharmaceutical compositions for administering iRNA and cyclodextrin are described in U.S. Pat. No. 7,427,605, the entire contents of which are incorporated herein by reference.

[0160] iRNA-encoding vector In another embodiment, TMPRSS6 gene-targeting iRNAs can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A. et al., TIG., 1996, vol. 12, pp. 5-10; Skillern, A. et al., WO 00 / 22113; Conrad, WO 00 / 22114; and U.S. Pat. No. 6,054,299). Expression can be transient (hours to weeks) or persistent (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, which can be integrative or non-integrative vectors. The transgene can also be constructed to allow it to be inherited as an extrachromosomal plasmid (Gassmann et al., Proceedings of the National Academy of Sciences, 1995, 92:1292).

[0161] Each iRNA strand or strands can be transcribed from the promoter on the expression vector.When expressing two separate strands to produce, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or infection).Alternatively, each of the promoters can be transcribed from the individual strands of dsRNA by being located on the same expression plasmid.In one embodiment, the dsRNA strands are expressed as inverted repeat polynucleotides that are linked by a linker polynucleotide sequence, so that the dsRNA has a stem-loop structure.

[0162] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting desired nucleic acid fragments. Delivery of iRNA expression vectors can be by systemic administration, such as intravenous or intramuscular administration, administration to target cells explanted from a patient and then reintroduced into the patient, or any other means that allows for introduction into desired target cells.

[0163] iRNA expression plasmids can be transfected into target cells as complexes with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown, targeting different regions of a target RNA over a period of one week or more, are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of cells in vitro can be ensured using markers that confer resistance to specific environmental factors (e.g., antibiotics and drugs) on transfected cells, such as hygromycin B resistance.

[0164] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may contain viral sequences for transfection, if desired. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure iRNA expression in target cells. Other contemplated aspects of vectors and constructs are described in more detail below.

[0165] Vectors useful for delivering iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulated / inducible expression.

[0166] iRNA expression can be precisely regulated using inducible regulatory sequences that are sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB Journal, 8:20-24). Suitable inducible expression systems for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art can select appropriate regulatory / promoter sequences based on the intended use of the iRNA transgene.

[0167] In certain embodiments, viral vectors containing nucleic acid sequences encoding iRNAs may be used. For example, retroviral vectors may be used (see Miller et al., Meth. Enzymol. 217:581-599, 1993). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding the iRNAs are cloned into one or more vectors, facilitating delivery of the nucleic acid to a patient. More details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302, 1994, which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells, for example, to generate stem cells that are more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are Clowes et al., J. Clin. Invest., 93:644-651 (1994); Kiem et al., Blood, 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy, 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel., 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Pat. No. 6,143,520; U.S. Pat. No. 5,665,557; and U.S. Pat. No. 5,981,276, which are incorporated herein by reference.

[0168] Adenoviruses are also being considered for use in delivering iRNA. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelia. Adenoviruses naturally infect respiratory epithelia, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development, Vol. 3, pp. 499-503, 1993, present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy, 5:3-10, 1994, demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science, Vol. 252, pp. 431-434 (1991); Rosenfeld et al., Cell, Vol. 68, pp. 143-155 (1992); Mastrangeli et al., J. Clin. Invest., Vol. 91, pp. 225-234 (1993); WO 94 / 12649; and Wang et al., Gene Therapy, Vol. 2, pp. 775-783 (1995). Suitable AV vectors for expressing the iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al., 2002, Nature. This is described in Nat. Biotech., Vol. 20, pp. 1006-1010.

[0169] The use of adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300, 1993; U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, e.g., with either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering vectors to target cells are described in Samulski R et al., 1987, J. Virol., 61, pp. 3096-3101; Fisher KJ et al., 1996, J. Virol., 70, pp. 520-532; Samulski R et al., 1989, J. Virol., 1999, The Journal of Virology, 2000, pp. 111-114, the disclosures of which are incorporated herein by reference in their entirety. J. Virol., vol. 63, pp. 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; WO 94 / 13788; and WO 93 / 24641.

[0170] Another preferred viral vector is a vaccinia virus, eg, an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, a poxvirus, eg, an avipox, such as fowlpox or canarypox.

[0171] The tropism of viral vectors can be modified, if desired, by pseudotyping the vector with envelope proteins or other surface antigens from other viruses or by substituting capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by expressing different capsid protein serotypes; see, for example, Rabinowitz JE et al., 2002, J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0172] The vector pharmaceutical preparation can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0173] III. iRNA-Containing Pharmaceutical Compositions In one embodiment, provided herein is a pharmaceutical composition containing an iRNA and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with the expression or activity of the TMPRSS6 gene, such as pathological processes mediated by TMPRSS6 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery, such as by intravenous (IV) delivery.

[0174] The pharmaceutical compositions provided herein are administered at a dosage sufficient to inhibit expression of the TMPRSS6 gene. Generally, suitable doses of iRNA range from 0.01 to 200.0 milligrams per kilogram of recipient body weight per day, generally from 1 to 50 mg per kilogram of body weight per day. For example, dsRNA may be administered at 0.05 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, or 50 mg / kg per single administration. Pharmaceutical compositions may be administered daily, weekly, monthly, or bimonthly. Alternatively, compositions may be administered twice weekly, twice monthly, or once every two, three, or four weeks. In some embodiments, iRNA is administered in two, three, or more subdoses at appropriate intervals throughout the day, or even via continuous infusion or delivery via a controlled-release formulation. In this case, the amount of iRNA contained in each partial dose must be correspondingly smaller to achieve the total daily dose.The dosage unit can also be formulated for delivery over several days, for example, by using a conventional sustained-release formulation that provides sustained release of iRNA over several days.Sustained-release formulations are well known in the art and are particularly useful for delivering active substances to specific sites, which can be used with the active substances of the present invention.In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0175] The effect of a single dose on TMPRSS6 levels may be prolonged, such that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart.

[0176] Those skilled in the art will understand that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other diseases present, may influence the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. Effective dosages and in vivo half-lives of the individual iRNAs encompassed by the invention may be estimated based on in vivo studies using appropriate animal models using conventional procedures or as described elsewhere herein.

[0177] Advances in mouse genetics have led to the creation of several mouse models for studying various human diseases, including pathological processes mediated by TMPRSS6 expression. These models can be used for in vivo testing of iRNAs and for determining therapeutically effective doses. A suitable mouse model is, for example, a mouse carrying a transgene that expresses human TMPRSS6.

[0178] The present invention also includes pharmaceutical compositions and formulations containing the iRNA compounds featured in the present invention. The pharmaceutical compositions of the present invention may be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be topical (e.g., via a transdermal patch), pulmonary, for example, via inhalation or insufflation of powders or aerosols, including nebulizers; intratracheal, intranasal, transepidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal administration, for example, via an implanted device; or intracranial administration, for example, intracerebral, intrathecal, or intraventricular.

[0179] The iRNA can be delivered in a manner that targets a specific tissue, such as the liver (e.g., liver parenchymal cells). Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is in admixture with a topical delivery agent, such as a lipid, liposome, fatty acid, fatty acid ester, steroid, chelating agent, or surfactant. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA featured in the present invention may be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNA may be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20 Topical formulations include, but are not limited to, alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0180] Liposomal formulation Besides microemulsions, which have been studied and used for drug formulation, there are many other organized surfactant structures. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted considerable attention from the perspective of drug delivery due to their specificity and the duration of action they offer. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayer group.

[0181] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse efficiently with the cell wall, but are taken up by macrophages in vivo.

[0182] To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. It is therefore desirable to use liposomes that are highly deformable and can pass through such pores.

[0183] Additional advantages of liposomes include the following: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; and liposomes can protect drugs encapsulated in their internal compartments from metabolism and degradation (Rosoff, Pharmaceutical Dosage Forms, edited by Lieberman, Rieger, and Banker, 1988, Vol. 1, p. 245, Marcel Dekker, Inc., New York, NY). Important considerations in the preparation of liposomal formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

[0184] Liposomes are useful for the transfer and delivery of active ingredients to the site of action.Because liposome membrane is structurally similar to biological membrane, when liposome is applied to tissue, liposome begins to integrate with cell membrane, and as liposome and cell fusion proceeds, liposome contents are released into cells, where active agents can act.

[0185] Liposomal formulations have been the focus of extensive research as a delivery mode for many drugs. Evidence is emerging that liposomes offer several advantages over other formulations for topical administration. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of both hydrophilic and hydrophobic drugs intradermally.

[0186] Several reports have detailed the ability of liposomes to deliver active substances, including high molecular weight DNA, into the skin. Compounds, including painkillers, antibodies, hormones, and high molecular weight DNA, have been administered to the skin. The majority of applications have resulted in targeting of the upper epidermis.

[0187] Liposomes are divided into two broad classes. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA / liposome complexes bind to the negatively charged cell surface and are internalized inside endosomes. The acidic pH within the endosome causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun. 1987, 147, 980-985).

[0188] pH-sensitive or negatively charged liposomes do not complex with nucleic acids but rather encapsulate them. Because both DNA and lipids have similar charges, repulsion rather than complexation occurs. Nevertheless, some DNA is encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19:269-274).

[0189] One major type of liposome composition contains phospholipids in addition to naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be made from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally made from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is made from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is made from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0190] Several studies have evaluated topical delivery of liposomal formulations to the skin. Application of interferon-containing liposomes to guinea pig skin resulted in a reduction in cutaneous herpes sores, whereas other means of interferon delivery (e.g., as a solution or emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, vol. 2, pp. 405-410). An additional study tested the effectiveness of administering interferon as part of a liposomal formulation compared with administering interferon using an aqueous system and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, vol. 18, pp. 259-265).

[0191] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their efficacy in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A into the dermis of mouse skin. The results suggested that such nonionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., STP Pharma. Sci., 1994, Vol. 4, No. 6, p. 466).

[0192] Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1 or (B) derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the improved circulation half-life of these sterically stabilized liposomes is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223:42; Wu et al., Cancer Research, 1993, 53:3765).

[0193] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al., Ann. NY Acad. Sci., 1987, 507, 64, describe monosialoganglioside G M1 reported the ability of galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated by Gabizon et al., Proceedings of the National Academy of Sciences, Vol. 85, p. 6949, 1988. U.S. Pat. No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G M1or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0194] Numerous liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods for their preparation, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describe a nonionic detergent containing a PEG moiety, 2C 1215Gdescribed liposomes comprising PEG or PEG-stearate. Illum et al., FEBS Lett., 1984, vol. 167, p. 79, noted that hydrophilic coating of polystyrene particles with polymeric glycols resulted in significantly improved blood half-lives. Synthetic phospholipids modified by the addition of carboxylic acid groups of polyalkylene glycols (e.g., PEG) were described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, vol. 268, p. 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significantly increased blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, vol. 1029, p. 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes bearing covalently bound PEG moieties on their exterior surfaces are described in EP 0 445 131 B1 and WO 90 / 04384 to Fisher. Liposomal compositions containing 1-20 mole percent PEG-derivatized PE, and methods for their use, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and EP 0 496 813 B1). Liposomes comprising several other lipid-polymer complexes are disclosed in WO 91 / 05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.), and WO 94 / 20073 (Zalipsky et al.).Liposomes comprising PEG-modified ceramide lipids are described in WO 96 / 10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes whose surfaces can be further derivatized with functional moieties.

[0195] Some liposomes comprising nucleic acids are known in the art. International Publication No. 96 / 40062 to Thierry et al. discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. Patent No. 5,264,221 to Tagawa et al. discloses protein-bound liposomes and asserts that the contents of such liposomes may include dsRNA. U.S. Patent No. 5,665,710 to Rahman et al. describes a specific method for encapsulating oligodeoxynucleotides in liposomes. International Publication No. 97 / 04787 to Love et al. discloses liposomes comprising raf gene-targeting dsRNA.

[0196] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets that are so highly deformable that they can easily penetrate pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to the shape of skin pores), self-repair, often reach their targets without fragmentation, and are often self-loading. To create transfersomes, a surface edge activator, usually a surfactant, can be added to a standard liposome composition. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0197] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and ranking the properties of the many different surfactant types, both natural and synthetic, is by use of the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying the different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0198] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used over a wide pH range. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0199] If surfactant molecule carries negative charge when dissolved or dispersed in water, surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylate, acyl amide of amino acid, sulfate esters such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate.The most important members of anionic surfactant class are alkyl sulfate and soap.

[0200] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0201] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

[0202] The use of surfactants in pharmaceuticals, formulations and emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, 1999). From "The Art of Electromagnetics," Marcel Dekker, Inc., New York, NY, 1988, p. 285.

[0203] nucleic acid lipid particles In one embodiment, the TMPRSS6 dsRNA featured in the present invention is fully encapsulated in a lipid formulation to form, for example, SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLP. As used herein, the term "SPLP" refers to nucleic acid-lipid particles comprising plasmid DNA encapsulated within lipid vesicles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid complexes). SNALP and SPLP exhibit long circulatory life following intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the site of administration), making them extremely useful for systemic applications. SPLPs include "pSPLPs" containing encapsulated condensing agent-nucleic acid complexes, as described in WO 00 / 03683. The particles of the present 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. Additionally, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and WO 96 / 40964.

[0204] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) ranges from 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.

[0205] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA). 1,2-Dilinoleyl-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or mixtures thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

[0206] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0207] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (molar percentages), with a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0208] Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-(N-maleiminomethyl)-2-(4-methyl-2-propanol), and dioleoyl-phosphatidylethanolamine-4-(N-maleiminomethyl)-2-(4-methyl-2-propanol). The lipid may be an anionic or neutral lipid, including, but not limited to, 16-O-monomethyl-2-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0209] The conjugated lipid that inhibits particle aggregation may be, for example, without limitation, a polyethylene glycol (PEG)-lipid, including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that inhibits particle aggregation may be 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particles.

[0210] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. LNP01 In one embodiment, lipid-dsRNA nanoparticles (i.e., LNP01 particles) can be produced using lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application No. 12 / 056,230, filed March 26, 2008, the entire contents of which are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml; PEG-ceramide C16, 100 mg / ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) to a final ethanol concentration of about 35-45% and a final sodium acetate concentration of about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, such as the Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0211] [ka] LNP01 formulations are described, for example, in WO 2008 / 042973, which is incorporated herein by reference.

[0212] Additional exemplary lipid-dsRNA formulations are as follows:

[0213] [Table 1]

[0214] [Table 2] DSPC: Distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-dimyristoylglycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000) Formulations comprising SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0215] XTC-containing formulations are described, for example, in U.S. Provisional Patent Application No. 61 / 148,366, filed January 29, 2009; U.S. Provisional Patent Application No. 61 / 156,851, filed March 2, 2009; U.S. Provisional Patent Application No. 61 / 228,373, filed July 24, 2009; U.S. Provisional Patent Application No. 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0216] MC3-containing formulations are described, for example, in U.S. Provisional Patent Application No. 61 / 244,834, filed September 22, 2009, U.S. Provisional Patent Application No. 61 / 185,800, filed June 10, 2009, and International Application No. PCT / US10 / 28224, filed June 10, 2010, which are incorporated herein by reference.

[0217] ALNY-100-containing formulations are described, for example, in International Application PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0218] C12-200-containing formulations are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0219] As used herein, the term "LNPXX" (where "XX" is a number) is also referred to herein as "AFXX." For example, LNP09 is also referred to as AF09, and LNP12 is also known as or referred to as AF12.

[0220] Synthesis of cationic lipids Any of the compounds, such as the cationic lipids used in the nucleic acid-lipid particles featured in the present invention, can be prepared by known organic synthesis techniques, including those methods described in more detail in the Examples. Unless otherwise indicated, all substituents are as defined below.

[0221] "Alkyl" means a straight-chain or branched, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

[0222] Alkenyl refers to the alkyl as defined above, containing at least one double bond between adjacent carbon atoms.Alkenyl includes both cis and trans isomers.Representative straight-chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, etc.

[0223] "Alkynyl" means any alkyl or alkenyl as defined above further containing at least one triple bond between adjacent carbons. Representative straight-chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

[0224] "Acyl" means any alkyl, alkenyl, or alkynyl, as defined below, where the carbon at the point of attachment is replaced by an oxo group. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

[0225] "Heterocycle" means a saturated, unsaturated, or aromatic 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocycle containing one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, including bicycles in which any of the heterocycles below are fused to a benzene ring; the nitrogen and sulfur heteroatoms may or may not be oxidized, and the nitrogen heteroatom may or may not be quaternized. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls, as defined below. Examples of heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0226] The terms "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted acyl," and "optionally substituted heterocycle" mean that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, the substituents include oxo, halogen, heterocycle, -CN, -OR. x , -NR x R y , -NR x C(=O)R y , -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x , and -SO n NR x Ry n is 0, 1 or 2; R x and R y are the same or different and independently represent hydrogen, alkyl, or heterocycle, and each of said alkyl and heterocycle substituents may be selected from one or more of oxo, halogen, —OH, —CN, alkyl, —OR x , heterocycle, -NR x R y , -NR x C(=O)R y , -NR x SO2R y , -C(=O)R x , -C(=O)OR x , -C(=O)NR x R y , -SO n R x , and -SO n NR x R y It may be further substituted by:

[0227] "Halogen" means fluoro, chloro, bromo, and iodo. In some embodiments, the methods featured in this invention may require the use of protecting groups. Protecting group procedures are well known to those skilled in the art (see, for example, "Protective Groups in Organic Synthesis," Green, TW et al., Wiley-Interscience, New York, NY, 1999). Briefly, in the context of this invention, a protecting group is any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during a specific reaction and then removed to expose the original functional group. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

[0228] Synthesis of Formula A In some embodiments, the nucleic acid-lipid particles featured in the present invention have Formula A:

[0229] [ka] (wherein R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be substituted or unsubstituted; R3 and R4 are independently lower alkyl; or R3 and R4 can be joined together to form a heterocycle, which may be substituted or unsubstituted). In some embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Generally, lipids of the above formula A can be prepared according to the following reaction schemes 1 or 2, in which all substituents are as defined above unless otherwise specified.

[0230] [ka] Lipid A, in which R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be substituted or unsubstituted, and R3 and R4 are independently lower alkyl, or R3 and R4 can be taken together to form a heterocycle, which may be substituted or unsubstituted, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 1 and 2 results in ketal 3. Treatment of ketal 3 with amine 4 yields lipids of Formula A. Lipids of Formula A can be converted to the corresponding ammonium salts with an organic salt of Formula 5, where X is an anionic counterion selected from halogens, hydroxides, phosphates, sulfates, and the like.

[0231] [ka] Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. Reaction of 6 and 7 provides ketone 1. Conversion of ketone 1 to the corresponding lipid of formula A is as described in Scheme 1.

[0232] Synthesis of MC3 DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid) was prepared as follows: A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed on a rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol / dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed to give a colorless oil (0.54 g).

[0233] Synthesis of ALNY-100 The synthesis of ketal 519 [ALNY-100] was carried out using Scheme 3 below.

[0234] [ka] Synthesis of 515 To a stirred suspension of LiAlH (3.74 g, 0.09852 mol) in 200 mL of anhydrous THF in a two-necked RBF (1 L) was slowly added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF at 0°C under a nitrogen atmosphere. After the addition was complete, the reaction mixture was warmed to room temperature and then heated to reflux for 4 h. The progress of the reaction was monitored by TLC. After the reaction was complete (by TLC), the mixture was cooled to 0°C and quenched by the careful addition of saturated NaSO solution. The reaction mixture was stirred at room temperature for 4 h and filtered. The residue was washed well with THF. The filtrate and washings were combined, diluted with 400 mL of dioxane and 26 mL of concentrated HCl, and stirred at room temperature for 20 min. Volatilities were stripped under vacuum to give the hydrochloride salt of 515 as a white solid. Yield: 7.12g 1H-NMR (DMSO, 400MHz): 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 To a stirred solution of compound 515 in 100 mL of dry DCM in a 250 mL two-necked RBF, NEt (37.2 mL, 0.2669 mol) was added and cooled to 0°C under a nitrogen atmosphere. After the slow addition of N-(benzyloxycarbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of dry DCM, the reaction mixture was allowed to warm to room temperature. After the reaction was complete (2-3 h by TLC), the mixture was washed successively with 1 N HCl solution (1 × 100 mL) and saturated NaHCO solution (1 × 50 mL). The organic layer was then dried over anhydrous NaSO, and the solvent was evaporated to give the crude product, which was purified by silica gel column chromatography to give 516 as a sticky mass. Yield: 11g (89%).1H-NMR (CDCl3, 400MHz): 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 Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of a 10:1 acetone / water mixture in a 500 mL one-neck RBF at room temperature, to which N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) was added, followed by 4.2 mL of a 7.6% solution of OsO (0.275 g, 0.00108 mol) in tert-butanol. After completion of the reaction (approximately 3 h), the mixture was quenched by the addition of solid NaSO, and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 × 100 mL), followed by saturated NaHCO (1 × 50 mL) solution, water (1 × 30 mL), and finally brine (1 × 50 mL). The organic phase was dried over anhydrous NaSO, and the solvent was removed in vacuo. Silica gel column chromatographic purification of the crude material gave a diastereomeric mixture which was separated by preparative HPLC. Yield: 6g crude 517A-Peak-1 (white solid), 5.13 g (96%). H-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+NH] - 283.5 present, HPLC - 97.86%. Stereochemistry confirmed by X-ray.

[0235] Synthesis of 518 Using a procedure similar to that described for the synthesis of compound 505, compound 518 was obtained as a colorless oil (1.2 g, 41%). H-NMR (CDCl, 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%. General procedure for the synthesis of compound 519 A solution of compound 518 (1 eq) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH (1 M, 2 eq) in THF. After the addition was complete, the mixture was heated at 40 °C for 0.5 h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous NaSO, then filtered through Celite and concentrated to an oil. Column chromatography afforded pure 519 as a colorless oil (1.3 g, 68%). C NMR = 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; Electrospray MS (+ve): Molecular weight calculated for C44H80NO2 (M+H)+, 654.6, found 654.6. Formulations prepared by either standard or non-extrusion methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be a whitish, translucent solution without aggregates or sediment. 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). Particle size should be approximately 20-300 nm, such as 40-100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation and encapsulated fraction was estimated using a dye exclusion assay. Samples of formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting detergent, such as 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the detergent-containing sample compared to a standard curve. Encapsulation fraction is calculated by subtracting "free" dsRNA content (measured by signal in the absence of surfactant) from total dsRNA content. The percentage of encapsulated dsRNA is typically >85%. In SNALP formulations, particle size is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm, and at least 120nm. Suitable ranges are typically at least about 50nm to at least about 110nm, at least about 60nm to at least about 100nm, or at least about 80nm to at least about 90nm.

[0236] Compositions and preparations for oral administration include powder or granule, microparticle, nanoparticle, suspension or solution in water or non-aqueous medium, capsule, gel capsule, sachet, tablet or mini-tablet.Thickener, flavoring agent, diluent, emulsifier, dispersing aid or binder may be required.In some embodiments, oral preparations are those in which the DsRNA of the present invention is administered in combination with one or more penetration-promoting surfactants and chelating agents.Suitable surfactants include fatty acid and / or ester or their salt, bile acid and / or their salt. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglyceride, diglyceride, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as fatty acid / salts combined with bile acids / salts. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA featured in the present invention can be orally delivered in granular form, including spray-dried particles, or can be complexed to form micro- or nanoparticles.DsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkylcyanoacrylates, DEAE-derivatized polyimines, pullulans, cellulose, and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexyl Examples of suitable dsRNA formulations include hydroxybenzoates, DEAE-acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0237] Compositions and formulations for parenteral, intraparenchymal (intracerebral), intrathecal, intraventricular, or intrahepatic administration may comprise sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0238] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. When treating liver disorders, such as liver cancer, liver-targeted formulations are particularly preferred.

[0239] The pharmaceutical preparations of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include combining the active ingredient with pharmaceutical carriers or excipients. Generally, the preparations are prepared by uniformly and intimately combining the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0240] The compositions of the present invention may be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may 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 carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0241] Additional formulations emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid in the form of droplets, usually greater than 0.1 μm in diameter (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; Idson, in Pharmaceutical Dosage Forms, edited by Lieberman, Rieger, and Banker, 1988, Marcel Dekker). Dekker, Inc., New York, NY, Vol. 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 2, p. 335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301.) Emulsions are often biphasic systems comprising two immiscible liquid phases that are intimately mixed and dispersed within one another.In general, emulsions may be either water-in-oil (w / o) or oil-in-water (o / w). When the aqueous phase is finely dispersed and dispersed as minute droplets in the bulk oily phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oily phase is finely dispersed and dispersed as minute droplets in the bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion. Emulsions may contain additional components in addition to the dispersed phase and the active agent, which may be present as a solution in either the aqueous or oily phase or as a separate phase itself. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions comprising more than two phases, such as in the case of oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o / w emulsion surround small water droplets constitute w / o / w emulsions. Similarly, oil droplet systems encapsulated in globules of water and stabilized within an oily continuous phase provide o / w / o emulsions.

[0242] Emulsions are characterized by little or no thermodynamic stability. Frequently, the dispersed or discontinuous phase of an emulsion is well dispersed within the external or continuous phase and is maintained in this form through the use of emulsifiers or formulation viscosity. Either of the emulsion phases may be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Another means of stabilizing emulsions involves the use of emulsifiers, which may be incorporated into either of the emulsion phases. Emulsifiers may be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (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; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 199).

[0243] Synthetic surfactants, also known as surface active agents, have a wide range of uses in emulsion formulations and are reviewed in the literature (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, Vol. 1, p. 285; Idson, Pharmaceutical Dosage Forms, (See, for example, "Surfactants in the Science of Surfactants," edited by Lieberman, Rieger, and Banker, 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 199.) Surfactants are typically amphiphilic, comprising hydrophilic and hydrophobic moieties. The ratio of hydrophilic to hydrophobic moieties is referred to as the surfactant's hydrophile / lipophile balance (HLB), and is a useful tool for classifying and selecting surfactants in formulation preparations.Surfactants may be classified into different classes based on the nature of the hydrophilic group: 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, Vol. 1, p. 285).

[0244] Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorbent bases with hydrophilic properties, such as anhydrous lanolin and hydrophilic petrolatum, can absorb water to form water-in-oil emulsions while still maintaining their semi-solid consistency. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, especially in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0245] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker, eds., 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker, eds., 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 199).

[0246] Hydrophilic colloids, or hydrocolloids, include natural gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around dispersed phase droplets and by increasing the viscosity of the external phase.

[0247] Emulsions often contain several components, such as carbohydrates, proteins, sterols, and phospholipids, which can easily support the growth of microorganisms, so preservatives are often incorporated into these preparations.The preservatives commonly used in emulsion preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.Antioxidants are also generally added to emulsion preparations to prevent the preparation from deteriorating.The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene; or reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0248] The application of emulsion formulations via dermal, oral, and parenteral routes and methods for their preparation have been reviewed in the literature (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; Idson, in Pharmaceutical Dosage Forms, edited by Lieberman, Rieger, and Banker, 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 199).Emulsion formulations for oral delivery are very widely used due to their ease of formulation and efficiency in terms of absorption and bioavailability (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; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245; Idson, Pharmaceutical Dosage Forms, (See, for example, "Emulsifiers and Forms," ​​edited by Lieberman, Rieger, and Banker, 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 199.) Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutrients are among the materials commonly administered orally as o / w emulsions.

[0249] In one embodiment of the present invention, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion may be defined as a system of water, oil, and an amphiphile that is a single, optically isotropic, and thermodynamically stable solution (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich, NG., and Ansel, HC., eds., 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, Vol. 1, p. 245). Typically, microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a transparent system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, "Controlled Release of Drugs: Polymers and Aggregate Systems," edited by Rosoff, M., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared through the combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte.Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0250] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with comprehensive knowledge of microemulsion formulation (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; Rosoff, Pharmaceutical Dosage Forms, pp. 111-114, 2004). (See, e.g., "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger, and Banker, eds., 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245; Block, "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger, and Banker, eds., 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 335.) Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs into a formulation of thermodynamically stable droplets that form spontaneously.

[0251] 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 pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. Cosurfactants, which are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase interfacial fluidity by penetrating the surfactant film, resulting in irregular coatings due to the gaps between surfactant molecules. However, microemulsions may be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, aqueous pharmaceutical solutions, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0252] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, pp. 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, pp. 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential drug absorption enhancement due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical (See, e.g., J. Pharmaceutical Research, 1994, Vol. 11, p. 1385; Ho et al., J. Pharm. Sci., 1996, Vol. 85, pp. 138-143). Microemulsions often form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or iRNA. Microemulsions have been effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improve local cellular uptake of iRNA and nucleic acids.

[0253] The microemulsions of the present invention may also contain additional ingredients and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve formulation properties and enhance absorption of the iRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0254] penetration enhancers In one embodiment, the present invention uses various penetration enhancers to achieve 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 lipid-soluble or lipophilic drugs can easily pass through cell membranes. It has been discovered that even non-lipophilic drugs can pass through cell membranes if the membrane they pass through is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

[0255] Penetration enhancers may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M., "Surfactants and polymers in drug delivery"). (See, e.g., "Drug Delivery Systems," Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92.) Each of the aforementioned classes of penetration enhancers is described in more detail below.

[0256] Surfactants: In the context of the present invention, surfactants (or "surface active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved iRNA absorption through mucous membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether) (see, e.g., Malmsten, M., Surfactants and Polymers in Drug Delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40, p. 252).

[0257] Fatty acids: Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1~20 Included are alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, e.g., Touitou, E. et al., Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carriers, vol. 1, pp. 111-115, 2006). Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, Vol. 7, pp. 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, Vol. 44, pp. 651-654).

[0258] Bile Salts: The physiological role of bile includes facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M., "Surfactants and Polymers in Drug Delivery"). (See Brunton, Chapter 38, "The Pharmacological Basis of Therapeutics," Informa Health Care, New York, NY, 2002; Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th ed., Hardman et al., eds., McGraw-Hill, New York, 1996, pp. 934-935.) A variety of naturally occurring bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE).(See, 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; Swinyard, Chapter 39, in Remington's Pharmaceutical Sciences, 18th ed., Gennaro, ed., Mack Publishing. Co., Easton, Pa., 1990, pp. 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, Vol. 7, pp. 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, Vol. 263, p. 25; Yamashita et al., J. Pharm. Sci., 1990, Vol. 79, pp. 579-583).

[0259] Chelating Agents: Chelating agents, as used in the context of the present invention, can be defined as compounds that form complexes with metal ions, thereby removing them from solution, resulting in improved iRNA absorption through mucosal membranes. With regard to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as deoxyribonuclease inhibitors, since most DNA nucleases require divalent metal ions for catalysis and are inhibited by chelating agents (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-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines). (See, e.g., 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, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, Vol. 7, pp. 1-33; Buur et al., J. Control Rel., 1990, Vol. 14, pp. 43-51).

[0260] Non-chelating non-surfactant: As used herein, a non-chelating non-surfactant penetration enhancer compound may be defined as a compound that demonstrates insignificant activity as a chelating agent or as a surfactant, yet still enhances absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, Vol. 7, pp. 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0261] Agents that enhance cellular uptake of iRNA may also be added to pharmaceuticals and other compositions of the present invention. For example, cationic lipids such as lipofectin (U.S. Patent No. 5,705,188 to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97 / 30731 to Lollo et al.) are also known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen;(Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass; aD1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™ / LipoGen™ (Invivogen; 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™ transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International;Examples of suitable anti-inflammatory drugs include B-Bridge International (Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA);

[0262] Other agents may be utilized to enhance the penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol; pyrroles such as 2-pyrrole; azone; and terpenes such as limonene and menthone.

[0263] Carrier Certain compositions of the present invention also incorporate a carrier compound into their formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or an analog thereof, that is inert (i.e., has no biological activity itself) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of nucleic acids and carrier compounds, typically in excess of the latter substance, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculatory reservoirs, likely due to competition between the carrier compound and the nucleic acid for their normal receptors. For example, recovery of partial phosphorothioate dsRNA in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev. 1995, 5, pp. 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev. 1996, 6, pp. 177-183).

[0264] excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected with the intended mode of administration in mind to provide the desired bulk, consistency, etc., when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (such as starch, sodium starch glycolate, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

[0265] The composition of the present invention can be prepared using pharmaceutically acceptable organic or inorganic excipients that do not adversely react with nucleic acid and are suitable for oral administration.Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone etc.

[0266] Preparations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of nucleic acids in liquid or solid oil bases.The solution may also contain buffers, diluents, and other suitable additives.Pharmaceutically acceptable organic or inorganic excipients suitable for oral administration that do not adversely affect nucleic acids may be used.

[0267] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0268] Other ingredients The compositions of the present invention may further contain other auxiliary ingredients conventionally found in pharmaceutical compositions at their technically established usage levels. Thus, for example, the compositions may contain additional compatible pharmacologically active ingredients, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, such materials should not excessively interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0269] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0270] In some embodiments, pharmaceutical compositions featured herein comprise (a) one or more iRNA compounds and (b) one or more anti-cytokine biologics that function via a non-RNAi mechanism. Examples of such biologics include biologics that target IL1β (e.g., anakinra), IL6 (e.g., tocilizumab), or TNF (e.g., etanercept, infliximab, adlimumab, or certolizumab).

[0271] The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

[0272] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. Dosages of the compositions featured herein generally fall within a range of circulating concentrations, including the ED50, with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods featured herein, a therapeutically effective dose can be initially estimated from cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range (e.g., achieve a reduction in polypeptide concentrations) of the compound, or polypeptide product of the target sequence, if appropriate, including the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0273] In addition to their administration as discussed above, the iRNAs described herein may be administered in combination with other known agents effective in treating pathological processes mediated by TMPRSS6 expression. In any event, the treating physician may adjust the amount and timing of iRNA administration based on the results observed using standard measures of efficacy known in the art or described herein.

[0274] Methods for treating diseases caused by TMPRSS6 gene expression The present invention particularly relates to the use of TMPRSS6-targeting iRNAs and compositions containing at least one such iRNA for treating TMPRSS6-mediated disorders or diseases. Compositions containing TMPRSS6-targeting iRNAs are used to treat disorders associated with elevated iron levels, such as thalassemia (e.g., beta-thalassemia intermedia or alpha-thalassemia), primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, sideroblastic anemia, hemolytic anemia, dyshematopoietic anemia, or sickle cell anemia. In one embodiment, TMPRSS6 iRNAs are used to treat hemoglobinopathies. The TMPRSS6 iRNAs featured in the present invention can also be used to treat elevated iron levels caused by other conditions, such as chronic alcoholism.

[0275] In thalassemia, the bone marrow synthesizes insufficient hemoglobin chains; this in turn reduces red blood cell production, causing anemia. Either the alpha or beta chains can be affected, but beta thalassemia is more common; newborns are healthy because their bodies still produce HbF, which lacks the beta chain; within the first few months of life, the bone marrow switches to producing HbA, and symptoms begin to appear.

[0276] β - Thalassemia is caused by the non-expression of the HBB gene (β 0 ) or low expression (β + ) resulting from a mutation in one of the alleles. - Thalassemia varies in severity depending on the genotype, and is classified as attenuated / trait beta - Thalassemia (β / β 0 or β / β+), intermediate β - Thalassemia (β 0 / β+), and severe β - Thalassemia (β 0 / β 0 or β + / β + ) is included.

[0277] While thalassemia intermedia (TI) typically exhibits minimal hemolysis, β-thalassemia major (TM) is typically associated with massive hemolysis, resulting in, for example, anemia and splenomegaly; and highly ineffective hematopoiesis, resulting in bone marrow drive (bone changes, osteopenia), increased erythropoietin synthesis, hepatosplenomegaly, hematopoietic drug consumption (megablastic anemia), and blood hyperuricemia. iRNAs featured in the present invention, such as TMPRSS6 iRNA, are typically used to treat iron overload (e.g., aβ thalassemia) associated with thalassemia, which is more TI-like. 0 / β+, β / β 0 or treatment of individuals with the β / β+ genotype.

[0278] Symptoms of beta thalassemia also include complications resulting from therapy, such as iron overload, which can cause endocrine disorders, liver fibrosis, and cardiac fibrosis. Administration of an iRNA agent that targets TMPRSS6 can be effective in treating one or more of these symptoms.

[0279] Alpha thalassemia is caused by non-expression of the HBA1 or HBA2 genes (α 0 ) or low expression (α + ) resulting from a mutation in one of the alleles. - Thalassemia varies in severity depending on the genotype, and can be classified as trait thalassemia (-α / αα), Hb Bart, and hydrops fetalis (α 0 / α 0 ), and alpha thalassemia minor (-- / αα), (-α / -α), and HbH disease (-- / -α). Fewer alpha-globin chains are produced, resulting in excess beta chains in adults and excess gamma chains in newborns. The excess beta chains form an unstable tetramer (called four beta chain hemoglobin H or HbH), which has an abnormal oxygen dissociation curve. Administration of an iRNA agent targeting TMPRSS6 can be effective in treating iron overload in subjects with alpha thalassemia.

[0280] Symptoms of hemochromatosis include, for example, abdominal pain, joint pain, fatigue, lack of energy, weakness, darkening of the skin (often referred to as "bronzing") and hair loss.Administering iRNA agents that target TMPRSS6 can be effective in treating one or more of these symptoms.

[0281] Other conditions associated with iron overload include increased risk of liver disease (cirrhosis, cancer), heart attack or heart failure, diabetes, osteoarthritis, osteoporosis, metabolic syndrome, hypothyroidism, hypogonadism, and, in some cases, premature death. Improper iron management resulting in 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 2, 3, or 4, can treat one or more of these conditions or prevent the onset or progression of diseases or disorders aggravated by increased iron levels.

[0282] The present invention further relates to the use of iRNAs or pharmaceutical compositions thereof in combination with other pharmaceuticals and / or other therapies, e.g., known pharmaceuticals and / or known therapies, e.g., those currently used to treat disorders associated with elevated iron levels. For example, in certain embodiments, TMPRSS6-targeting iRNAs are administered in combination with, e.g., iron chelators (e.g., desferoxamine), folic acid, blood transfusions, phlebotomy, ulcer management drugs, drugs for increasing fetal hemoglobin levels (e.g., hydroxyurea), infection suppressants (e.g., antibiotics and antivirals), drugs for treating thrombotic conditions, or stem cell or bone marrow transplantation. Stem cell transplantation may utilize stem cells from the umbilical cord of a related person, e.g., a sibling. Exemplary iron chelators include desferoxamine, Deferasirox (Exjade), deferoprone, vitamin E, wheat germ oil, tocophersolan, and indicaxanthin.

[0283] The iRNA and the additional therapeutic agent can be administered in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein. Administration of the TMPRSS6 iRNA and the additional therapeutic agent can be simultaneous or at different times, and in any order.

[0284] The present invention features a method for administering a TMPRSS6-targeting iRNA agent to a patient with a disease or disorder mediated by TMPRSS6 expression, such as a disorder associated with elevated iron levels. Administration of dsRNA can reduce iron levels, reduce ferritin levels, and / or reduce transferrin saturation levels. For example, administration of dsRNA can reduce serum iron levels and / or reduce serum ferritin levels. Transferrin saturation levels can be reduced by 5%, 10%, 15%, 20%, 25% or more. Transferrin saturation levels can be reduced to less than 50%, less than 45%, less than 40%, less than 35%, or less than 35%. Transferrin saturation is a measure of the amount of serum transferrin bound to iron and corresponds to the ratio of serum iron to total iron binding capacity.

[0285] "Reduce" in this context means a statistically significant decrease in such levels, which can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, preferably to a level recognized as within the normal range for individuals with such disorders.

[0286] The effectiveness of disease treatment or prevention can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the drug dose required to maintain therapeutic effect, the level of a disease marker, or the level of any other measurable parameter appropriate for the given disease being treated or prevented. It is well within the capabilities of those skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one of these parameters or any combination of parameters. For example, the effectiveness of a given treatment regimen can be monitored by monitoring the level of transferrin saturation or serum ferritin.

[0287] Iron level tests are typically performed on a patient's blood sample. Iron level tests measure the amount of serum iron carried by the protein transferrin. The TIBC (Total Iron-Binding Capacity) test measures the amount of iron the blood would carry if transferrin were fully saturated. Because transferrin is produced by the liver, TIBC can be used to monitor liver function and nutrition. The transferrin test is a direct measurement of blood transferrin (also called siderophilin) ​​levels. Transferrin saturation levels can be calculated by dividing the serum iron level by the TIBC. The ferritin test measures the level of a blood protein that stores iron for later use by the body.

[0288] The iRNA treatments described herein can be used to treat individuals with elevated iron levels, which may be indicated by serum iron levels, such as iron levels measured at greater than 350 μg / dL, greater than 500 μg / dL, or greater than 1000 μg / dL. In one embodiment, the elevated serum iron level is, for example, greater than 15, 20, 25, or 30 mg / g dry weight.

[0289] The iRNA therapies described herein may be used to treat individuals with elevated iron levels, which may be indicated by elevated serum ferritin levels, such as ferritin levels measured at greater than 300 μg / L, greater than 500 μg / L, greater than 1000 μg / L, greater than 1500 μg / L, greater than 2000 μg / L, greater than 2500 μg / L, or greater than 3000 μg / L.

[0290] The iRNA treatments described herein can be used to treat individuals with elevated iron levels, which may be indicated by elevated serum transferrin levels, such as transferrin levels measured above 400 mg / dL, above 500 mg / L, or above 1000 mg / dL.

[0291] The iRNA treatments described herein can be used to treat individuals with moderately elevated iron levels, which may be indicated by a moderate increase in transferrin saturation levels, such as 40%, 45%, or 50% or greater saturation levels. In addition, the treatments described herein can be used to prevent elevated iron levels in individuals with only a slight increase in transferrin saturation. One skilled in the art can easily monitor transferrin saturation levels in subjects treated with iRNAs as described herein and assay for at least a 5% or 10% decrease in transferrin saturation levels.

[0292] The iRNA treatments described herein can be used to treat individuals with elevated iron levels, which may be indicated by TIBC values ​​greater than 400 μg / dL, greater than 500 μg / dL, or greater than 1000 μg / dL.

[0293] In some embodiments, the individual in need of treatment with TMPRSS6 siRNA has reduced hematocrit levels, reduced hemoglobin levels, increased red blood cell distribution width, increased reticulocytosis, reduced mature red blood cell count, increased unsaturated iron binding capacity, reduced ineffective hematopoiesis, reduced extramedullary hematopoiesis, and / or reduced HAMP1 expression levels.

[0294] Patients may be further monitored by assays of blood sugar (glucose) or alpha-fetoprotein levels, echocardiograms (e.g., to check heart function), electrocardiograms (ECGs) (e.g., to look at the electrical activity of the heart), imaging tests (such as CT scans, MRIs, and ultrasounds), and liver function tests. Excess iron staining or iron concentration may be measured in liver biopsy samples or to ascertain the degree of liver damage, e.g., the stage of liver disease.

[0295] A therapeutic or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is a lack of an otherwise expected worsening or onset of symptoms. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease may indicate effective treatment. The efficacy of a given iRNA agent or combination of agents may also be determined using an experimental animal model for a given disease known in the art. When using an experimental animal model, the efficacy of treatment is demonstrated when a statistically significant reduction in a marker or symptom is observed.

[0296] Alternatively, efficacy may be measured by a reduction in disease severity, as determined by one skilled in the art of diagnosis based on clinically accepted disease severity rating scales. A patient may be administered a therapeutic dose of iRNA, such as 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 2.5 mg / kg of dsRNA. The iRNA may be administered by intravenous infusion over a period of time, such as over 5, 10, 15, 20, or 25 minutes. Administration may be repeated periodically, for example, every other week (i.e., every two weeks) for one, two, three, four, or more months. After the initial treatment regimen, treatment may be administered less frequently. For example, after three months of biweekly administration, administration may be repeated once a month for six months or a year or more. Administration of iRNA can, for example, reduce TMPRSS6 levels in a patient's cells, tissues, blood, urine, or other compartment by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0297] Prior to administration of the full dose of iRNA, the patient may be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as allergic reactions or worsening symptoms. In another example, the patient may be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-α or INF-α) levels.

[0298] Many disorders associated with elevated iron levels are hereditary. Therefore, patients who require TMPRSS6 iRNA may be identified by taking a family history. Before prescribing or administering TMPRSS6 dsRNA, a healthcare provider such as a doctor or nurse, or a family member may take a family history. DNA testing may also be performed on patients to identify mutations in the TMPRSS6 gene before administering TMPRSS6 dsRNA to the patient. For example, a diagnosis of hereditary hemochromatosis may be confirmed by identifying two HFE (hemochromatosis) gene mutations, C282Y and H63D, listed in GenBank accession number CAB07442.1 (GI:1890180, recorded on October 23, 2008).

[0299] Due to the inhibitory effect on TMPRSS6 expression, the composition according to the present invention or a pharmaceutical composition prepared therefrom may improve the quality of life. Methods for regulating expression of the TMPRSS6 gene In yet another aspect, the invention provides a method for modulating (eg, inhibiting or activating) expression of the TMPRSS6 gene in a mammal.

[0300] In one embodiment, the method includes administering a composition featured in the present invention to a mammal to reduce expression of the target TMPRSS6 gene over an extended period of time, e.g., at least 2, 3, or 4 days or more, e.g., 1, 2, 3, or 4 weeks or more. The effect of reducing the target TMPRSS6 gene preferably results in reduced iron absorption and / or mobilization in the body. Reduced iron absorption or mobilization may be manifested by an observed decrease in serum ferritin levels, serum or liver iron levels, and / or serum transferrin saturation levels. In some embodiments, one or more of serum ferritin levels, serum or liver iron levels, or serum transferrin saturation levels are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more compared to pre-treatment levels. In some embodiments, serum ferritin levels are reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more compared to pre-treatment levels.

[0301] In another embodiment, the method includes administering a composition described herein to a mammal such that expression of the target TMPRSS6 gene is increased, e.g., by at least 10%, compared to an untreated animal. In some embodiments, activation of TMPRSS6 occurs over an extended period, e.g., at least 2, 3, 4 days or more, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks or more. Without wishing to be bound by theory, iRNAs may activate TMPRSS6 expression by stabilizing TMPRSS6 mRNA transcripts, interacting with promoters in the genome, and / or inhibiting inhibitors of TMPRSS6 expression.

[0302] iRNAs useful in the methods and compositions featured herein specifically target the RNA (primary or processed) of the target TMPRSS6 gene. Compositions and methods for inhibiting expression of these TMPRSS6 genes using iRNAs can be made and performed as described elsewhere herein.

[0303] In one embodiment, the method includes administering a composition containing an iRNA, wherein the iRNA comprises a nucleotide sequence complementary to at least a portion of a TMPRSS6 gene RNA transcript of the mammal being treated. When the organism being treated is a human, such as a mammal, the composition may be administered by any means known in the art, including, but not limited to, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, intratracheal (aerosol), intranasal, intrarectal, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection.

[0304] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the iRNA and methods featured in this invention, and suitable methods and materials are listed below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, shall prevail.In addition, materials, methods and examples are intended to be illustrative only and are not intended to be limiting.

[0305] (Example) Example 1. Interfering RNA (iRNA) synthesis Reagent Suppliers Unless the source of a reagent is specifically indicated herein, such reagents may be obtained from any supplier of molecular biology reagents of quality / purity standards for molecular biology applications.

[0306] Oligonucleotide synthesis Applicants have used several different methods to create the iRNA molecules described herein. This example describes one approach used. Those skilled in the art can prepare iRNAs as described herein using any method known in the art.

[0307] Oligonucleotides are synthesized on an AKTA oligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 50 Å, Prime Synthesis) is used. Synthesis) and RNA phosphoramidites with standard protecting groups, such as 5'-O-dimethoxytrityl-N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite (Pierce Pierce Nucleic Acids Technologies was used for oligonucleotide synthesis. 2'-F phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-ON,N'-diisopropyl-2-cyanoethyl-phosphoramidite, and 5'-O-dimethoxytrityl-2'-fluro-uridine-3'-ON,N'-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from Promega. All phosphoramidites were used at a concentration of 0.2 M in acetonitrile (CH3CN), except for guanosine, which was used at a concentration of 0.2 M in 10% THF / ANC (v / v). A 16-minute coupling / recycle time was used. The activating agent is 5-ethylthiotetrazole (0.75 M, American International Chemicals); iodine / water / pyridine is used for PO-oxidation, and PADS (2%) in 2,6-lutidine / ACN (1:1 v / v) is used for PS-oxidation.

[0308] The 3'-ligand conjugate chains are synthesized using a solid support containing the corresponding ligand. The introduction of a cholesterol unit into the sequence is carried out, for example, from hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate bond to obtain a hydroxyprolinol-cholesterol moiety. 5'-terminal Cy-3 and Cy-5.5 (fluorophore)-labeled iRNAs are available from Biosearch Technologies. The oligonucleotides are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidites purchased from AbbVie Technologies. Covalent attachment of ligands to the 5'-terminus and / or internal positions is achieved by using appropriately protected ligand-phosphoramidite building blocks. A 0.1 M solution of the phosphoramidite in anhydrous CH3CN is coupled to the solid support-bound oligonucleotide over a 15-minute period in the presence of 5-(ethylthio)-1H-tetrazole activator. Oxidation of the internucleotide phosphites to phosphates is reported (1) using standard iodine-water or by treating the conjugated oligonucleotide with tert-butyl hydroperoxide / acetonitrile / water (10:87:3) with a 10-minute oxidation wait time. Phosphorothioates are introduced by oxidation of the phosphites to phosphorothioates using sulfur transfer reagents such as DDTT (purchased from AM Chemicals), PADS, and / or Beaucage reagent. Cholesterol phosphoramidite was synthesized in-house and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterol phosphoramidite is 16 minutes.

[0309] Deprotection I (nucleobase deprotection) After synthesis is complete, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support by simultaneous deprotection of the base and phosphate groups using 80 mL of ethanolic ammonia [ammonia:ethanol (3:1)] mixture at 55 °C for 6.5 h. The bottle is briefly cooled on ice, and then the ethanolic ammonia mixture is filtered into a new 250 mL bottle. The CPG is washed with 2 × 40 mL volumes of ethanol / water (1:1 v / v). The volume of the mixture is then reduced to approximately 30 mL by roto-vap. The mixture is then frozen on dry ice and vacuum dried on a speed vac.

[0310] Deprotection II (removal of 2'-TBDMS group) The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA·3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60 °C for 90 min to remove the butyldimethylsilyl (TBDMS) group at the 2' position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. The oligonucleotides are stored in a freezer until purification.

[0311] analysis Oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification, with the choice of buffer and column depending on the sequence and / or the nature of the binding ligand.

[0312] HPLC purification Ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. Unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in-house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (Buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1 M NaBr (Buffer B). Full-length oligonucleotide-containing fractions are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotide is diluted to 150 μL with water and then pipetted into specialized vials for CGE and LC / MS analysis. The compounds are then analyzed by LC-ESMS and CGE.

[0313] iRNA preparation For a typical preparation of iRNA, equimolar amounts of sense and antisense strands are heated to 95°C for 5 minutes in 1x PBS and allowed to cool slowly to room temperature. The integrity of the duplex is confirmed by HPLC analysis.

[0314] The nucleic acid sequences are set forth below using standard nomenclature, particularly the abbreviations in Table 1. Table 1: Nucleotide monomer abbreviations used in the representation of nucleic acid sequences. These monomers, when present in an oligonucleotide, are understood to be linked to each other by 5'-3'-phosphodiester bonds.

[0315] [Table 3] Example 2. TMPRSS6 siRNA design Transcripts TMPRSS6-targeting siRNA was designed and synthesized using the human transcript NM_153609.2 (SEQ ID NO: 1, Figure 1) from the NCBI Refseq collection.

[0316] The siRNA duplex was designed with 100% identity to the TMPRSS6 gene. A total of 655 sense and 655 antisense human TMPRSS6-derived siRNA oligos were designed. The oligos are presented in Table 2. Additional sense and antisense human TMPRSS6-derived siRNA oligos are presented in Table 3. Modified sense and antisense human TMPRSS6-derived siRNA oligos are presented in Table 4.

[0317] Table 2. Sense and antisense strand sequences of human TMPRSS6 dsRNA

[0318] [Table 4]

[0319] [Table 5]

[0320] [Table 6]

[0321] [Table 7]

[0322] [Table 8] Table 3. Unmodified sense and antisense strand sequences of human TMPRSS6 dsRNA

[0323] [Table 9]

[0324] [Table 10]

[0325] [Table 11] Table 4. Modified sense and antisense strand sequences of human TMPRSS6 dsRNA

[0326] [Table 12]

[0327] [Table 13]

[0328] [Table 14] Synthesis of TMPRSS6 sequence TMPRSS6 iRNA sequences can be synthesized on a MerMade 192 synthesizer at the 1 μmole scale.

[0329] Endolight chemistry may be applied, as detailed below. All pyrimidines (cytosines and uridines) in the sense strand contained 2'-O-methyl bases (2'-O-methyl C and 2'-O-methyl U).

[0330] In the antisense strand, pyrimidines adjacent to ribo A nucleosides (towards the 5' position) can be replaced with their corresponding 2-O-methyl nucleosides. A two-base dTsdT extension can be introduced at the 3' end of both the sense and antisense sequences.

[0331] The sequence file can be converted to a text file to make it suitable for loading into MerMade 192 synthesis software. Synthesis, cleavage, and deprotection Synthesis of the TMPRSS6 sequence used solid-support oligonucleotide synthesis using phosphoramidite chemistry.

[0332] Synthesis of the above sequence can be carried out on a 1 μm scale in 96-well plates. Amidite solutions can be prepared at 0.1 M concentration, and ethylthiotetrazole (0.6 M in acetonitrile) can be used as the activating agent.

[0333] Synthetic sequences can be cleaved and deprotected in 96-well plates using methylamine in the first step and fluoride reagent in the second step. Crude sequences can be precipitated using an acetone:ethanol (80:20) mixture, and the pellet can be resuspended in 0.02 M sodium acetate buffer. Samples from each sequence can be analyzed by LC-MS to confirm identity and quantified by UV. Selected sample sets can also be analyzed by IEX chromatography to determine purity.

[0334] Purification and desalting All sequences can be purified on an AKTA explorer purification system using a Source 15Q column. Sample injection and collection can be performed in a 96-well (1.8 mL deep well) plate. A single peak corresponding to the full-length sequence can be collected in the eluent. The purified sequence can be desalted on a Sephadex G25 column using an AKTA purifier. The desalted TMPRSS6 sequence can be analyzed for concentration (by UV measurement at A260) and purity (by ion-exchange HPLC). Single strands can then be used for annealing.

[0335] Example 3. In vitro screening of TMPRSS6 siRNA duplexes for TMPRSS6 knockdown activity TMPRSS6 siRNA duplexes were screened for their ability to knock down TMPRSS6 expression in vitro, using single-dose screening, dose-response screening, and assessing host cell viability.

[0336] In vitro screening: Cell culture and transfection for single dose and dose response studies: HeLa or Hep3B cells (ATCC, Manassas, VA) were grown to near confluence in ATCC medium supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C in a 5% CO atmosphere and then released from the plate by trypsinization. Transfection was performed in 96-well plates by adding 5 μl of siRNA duplex per well to 14.8 μl of Opti-MEM + 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA; catalog number 13778-150) per well and incubating at room temperature for 15 minutes. Approximately 2 × 10 4Eighty microliters of complete growth medium without antibiotics containing HeLa or Hep3B cells was added to the siRNA mixture. Cells were cultured for either 24 or 120 hours prior to RNA purification. Single-dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose-response experiments were performed at final duplex concentrations of 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM.

[0337] Total RNA isolation using DYNABEADS® mRNA isolation kit (Invitrogen, part number: 610-12): The cells were collected and lysed in 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf® Thermomixer (the mixing speed was the same throughout the procedure). Ten microliters of magnetic beads and 80 μl of lysis / binding buffer mixture were added to a round-bottom plate and mixed for 1 minute. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were recaptured and the supernatant was removed. The beads were then washed with 150 μl of wash buffer B, captured, and the supernatant was removed. The beads were then washed with 150 μl of elution buffer, captured, and the supernatant was removed. The beads were then dried for 2 minutes. After drying, 50 μl of elution buffer was added and mixed for 5 minutes at 70°C. The beads were captured on a magnet for 5 minutes. 40 μl of the supernatant was removed and placed in another 96-well plate.

[0338] cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat. No. 4368813): A master mix of 2 μl of 10× buffer, 0.8 μl of 25× dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl of H O was added to 10 μl of total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 s, and a 4°C hold.

[0339] Real-time PCR: In fifty 384-well plates, 2 μl of cDNA was added per well to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Applied Biosystems; catalog number 4326317E), 0.5 μl of TMPRSS6 TaqMan Probe (Applied Biosystems; catalog number Hs00542184_m1), and 5 μl of Lightcycler 480 Probe Master Mix (Roche; catalog number 04887301001) (Roche; catalog number 04887301001). Real-time PCR was performed in an ABI7900HT Real-Time PCR System (Applied Biosystems) using the ΔΔCt (RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate unless otherwise noted in the summary table.

[0340] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with 10 nM AD-1955-transfected or mock-transfected cells. A four-parameter fit model using XLFit was used to calculate IC50s, normalized to AD-1955-transfected cells or to the lowest dose across the same dose range.

[0341] Viability screening. HeLa or Hep3B cells (ATCC, Manassas, VA) were grown to near confluence in ATCC medium supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C in a 5% CO atmosphere and then released from the plate by trypsinization. Cell viability was measured in HeLa and Hep3B cells on days 3 and 5 following transfection with 100, 10, 1, 0.1, 0.01, and 0.0001 nM siRNA. 2.5 x 10 cells were cultured per well in a 96-well plate. 3 ~5×10 3 Cells were seeded at a density of 1000 x 1000 cells. Each siRNA was assayed in triplicate, and the data were averaged. PLK1 and AD-19200-targeting siRNAs were included as positive controls for viability reduction, and AD-1955 as a negative control. PLK1 and AD-19200 cause a dose-dependent reduction in viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of a 96-well plate after 3 and 5 days and incubated at 37°C for 2 hours. 560 μl of CellTiter Blue (Promega) was then added to each well of a 96-well plate after 3 and 5 days and incubated at 37°C for 2 hours. Ex / 590 Em Plates were read in a spectrophotometer (Molecular Devices) at 100°C. Viability was expressed as the mean value of light units from three replicate transfections + / - the standard deviation.

[0342] In vitro knockdown of TMPRSS6 expression by TMPRSS6 siRNA duplexes Table 5 presents data showing TMPRSS6 knockdown in Hep3B cells transfected with TMPRSS6-targeting siRNA. Data are expressed as the percentage of TMPRSS6 message remaining in cells transfected with TMPRSS6-targeting siRNA compared to cells transfected with a negative control siRNA, AD-1955. Untreated cells ("naive" cells) served as a second negative control. All siRNAs were tested at least twice, and qPCR reactions were also performed in duplicate. Single-dose experiments were performed at final siRNA duplex concentrations of 10 nM and 0.1 nM.

[0343] Table 5. TMPRSS6 expression in ex vivo single dose screening

[0344] [Table 15]

[0345] [Table 16]

[0346] [Table 17] IC of selected TMPRSS6 siRNA duplexes in in vitro dose-response screening 50 Table 6 shows the IC of select TMPRSS6 siRNA duplexes determined from in vitro dose-response screening. 50Values ​​are presented. TMPRSS6 siRNA duplexes effective in the 10 nM and 0.1 nM single-dose screen (Table 5) were tested for TMPRSS6 knockdown activity in a dose response in Hep3B cells on days 1 and 5 following transfection. Dose-response experiments were performed at final siRNA duplex concentrations of 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM. For normalization, TMPRSS6 knockdown was measured relative to the nontargeting control AD-1955 or the value obtained at the lowest siRNA concentration of each duplex tested.

[0347] Table 6. IC of selected TMPRSS6 siRNA duplexes in in vitro dose-response screening 50

[0348] [Table 18] In vitro viability screening of HeLa and HEP3B cell lines transfected with TMPRSS6 siRNA duplexes Table 7 presents viability data for HeLa and HEP3B cell lines transfected with TMPRSS6 siRNA duplexes. Viability data are expressed as mean raw fluorescence units, where smaller values ​​represent decreased viability. Errors are expressed as standard deviations from three replicate transfections.

[0349] Table 7. Viability of HeLa and HEP3B cell lines transfected with TMPRSS6 siRNA duplexes.

[0350] [Table 19]

[0351] [Table 20]

[0352] [Table 21]

[0353] [Table 22] Example 4. Manual selection of TMPRSS6 siRNA duplexes To select specific TMPRSS6 siRNAs for use in further in vivo experiments, chemically modified siRNAs were transfected into HEP3B human liver tumor cells and screened for TMPRSS6 gene silencing activity. Two highly potent siRNAs with minimal predicted off-target potential and broad species reactivity, including human, cynomolgus monkey, rat, and mouse, were selected for in vivo evaluation. The efficacy of the two selected TMPRSS6 siRNAs was also confirmed in primary mouse hepatocytes. TMPRSS6 siRNA-1 (AD-46273) and TMPRSS6 siRNA-2 (AD-46286) both demonstrated potent TMPRSS6 gene silencing activity, with TMPRSS6 siRNA-1 (AD-46273) demonstrating an IC of 70 pM. 50 (Figure 2A), and TMPRSS6 siRNA-2 (AD-46286) had an IC of 140 pM. 50 was shown (Figure 2B).

[0354] Example 5. Effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 in WT C57BL / 6 mice Effect of TMPRSS6 siRNA on TMPRSS6 and HAMP1 mRNA expression in WT C57BL / 6 mice To evaluate the in vivo effects of LNP-TMPRSS6 siRNA-1 (AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286), 8-week-old female WT C57BL / 6 mice were administered 1 mg / kg of LNP-TMPRSS6 siRNA-1 (AD-46273), LNP-TMPRSS6 siRNA-2 (AD-46286), or LNP-AD-19551 (a non-mammalian luciferase-targeting siRNA) via tail vein IV injection. TMPRSS6 siRNA was formulated with LNP11 (MC3). 24 hours after administration, the mice were sacrificed, and their livers were removed, flash-frozen, and ground into powder. A small amount (approximately 20 mg) of liver powder was disrupted in lysis buffer and used for mRNA analysis by TaqMan®. A total of five mice were used per group. Data are expressed as a percentage of the LNP-Luc control ratio of target TMPRSS6 mRNA compared to β-actin mRNA. As shown in Figure 3A, there was specific and potent dose-dependent inhibition of hepatic TMPRSS6 mRNA expression by LNP-TMPRSS6 siRNA-1 (AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286) (data represent the mean ± standard deviation), with an ED of 0.035 mg / kg, respectively. 50 , and an ED of 0.18 mg / kg 50 As shown in Figure 3B, there was also a dose-dependent inhibition of hepatic HAMP1 mRNA expression by LNP-TMPRSS6 siRNA-1 (AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286).

[0355] Duration of TMPRSS6 siRNA-mediated silencing of TMPRSS6 and HAMP1 gene expression in WT C57BL / 6 mice To assess the duration of TMPRSS6 siRNA-mediated knockdown of TMPRSS6 and HAMP1 gene expression, 8-week-old WT C57BL / 6 mice were administered a single 1 mg / kg dose of LNP-TMPRSS6 siRNA-1 (AD-46273), LNP-Luc control (LNP-AD-1955), or PBS via tail vein IV injection; all siRNAs were delivered in the LNP11 formulation. Mice were sacrificed at 6 hours, 24 hours, 48 ​​hours, 3 days, 7 days, and 14 days. TMPRSS6 and HAMP1 mRNA expression levels in the liver were analyzed using TaqMan® assays and normalized to β-actin. Five mice were used per group, and data are presented in Figure 4 as mean values ​​+ / - standard deviation. As shown in Figure 4, a single 1 mg / kg dose of LNP-TMPRSS6 siRNA-1 (AD-46273) knocked down TMPRSS6 mRNA expression as early as 6 hours post-administration and reduced TMPRSS6 mRNA expression to approximately 90% of LNP-Luc or PBS controls over a 2-week period. HAMP1 gene expression increased beginning 24 hours post-administration, was maintained throughout the 2-week period, and reached a maximum of 200% of control levels on day 14 post-administration (Figure 4). Additionally, serum iron levels were assayed as a percentage of transferrin (Tf) saturation using an Olympus AU 400. Transferrin saturation levels were calculated as the ratio of serum iron to total iron-binding capacity (TIBC) and expressed as a percentage of transferrin saturation. The percentage of transferrin saturation decreased to approximately 50% beginning 24 hours post-administration and was maintained over a 2-week period, suggesting reduced circulating iron levels in serum (Figure 4). Level of TMPRSS6 siRNA-mediated TMPRSS6 silencing required to maintain TMPRSS6 siRNA-mediated effects on HAMP1 gene expression and serum iron levels in WT C57BL / 6 mice.

[0356] To assess the level of TMPRSS6 siRNA-mediated TMPRSS6 silencing required to maintain the TMPRSS6 siRNA-mediated effects on HAMP1 gene expression and serum iron levels in WT C57BL / 6 mice, C57BL / 6 mice were administered 0.3 mg / kg of LNP-TMPRSS6 siRNA-1 (AD-46273), LNP-Luc control, or PBS; all siRNAs were delivered in the LNP11 formulation. Mice were sacrificed at 5 hours, 24 hours, 48 ​​hours, 3 days, 7 days, 14 days, 21 days, and 28 days after administration. TMPRSS6 and HAMP1 mRNA expression levels were analyzed using TaqMan® assays and normalized to β-actin. Five mice were used per group, and data are presented in Figure 5 as mean values ​​+ / - standard deviation. As shown in Figure 5, a 90% maximum reduction in TMPRSS6 gene expression was achieved 24 hours after administration and maintained for up to 3 days after administration. By day 7 after treatment, TMPRSS6 gene expression was reduced by approximately 85%; HAMP1 gene expression was induced to approximately 250% of control; and transferrin saturation (%) was reduced by approximately 50% (Figure 5). By day 21 after treatment, TMPRSS6 gene expression was reduced by approximately 40%; HAMP1 gene expression was normalized; and serum iron levels, as measured by transferrin saturation (%), began to return to normal levels (Figure 5). In summary, maximal knockdown of TMPRSS6 mRNA expression was achieved at 24 hours after treatment and returned to approximately 50% of normal expression levels by 3 weeks after treatment; hepcidin mRNA levels increased as early as 24 hours and were maintained for up to 7 days after treatment; hepcidin levels returned to control levels by day 14 after treatment; and transferrin saturation, as an indicator of circulating iron levels, was reduced to 50% of normal levels by 24 hours after treatment and normalized toward week 4. Thus, the data presented in Figure 5 indicate that greater than 50% TMPRSS6 silencing is required to maintain the LNP-TMPRSS6 siRNA-1 (AD-46273)-mediated effects on HAMP1 gene expression and serum iron levels.

[0357] Effects of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hematological parameters in WT C57BL / 6 mice To evaluate the effects of TMPRSS6 siRNA-mediated silencing on hematological parameters, including hemoglobin (HGB) and hematocrit, WT C57BL / 6 mice were administered a single 1 mg / kg dose of TMPRSS6 siRNA-1 (AD-46273), LNP-Luc control, or PBS and subsequently sacrificed at different time points up to 2 weeks post-treatment. Hematological parameters, including hemoglobin (HGB), hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and reticulocyte hemoglobin content (Chr), were assayed using an Advia 120 analyzer. As shown in Figures 6A and 6B, silencing TMPRSS6 in Th3 / + mice resulted in reduced HGB (Figure 6A) and hematocrit (Figure 6B) in WT C57BL / 6 mice, with similar effects on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and reticulocyte hemoglobin content (Chr).

[0358] Example 6. Effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 in thalassemia mice (Th3 / +) Effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on serum iron parameters in thalassemia mice (Th3 / +) To evaluate the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on serum iron parameters, including iron levels, unsaturated iron-binding capacity (UIBC), and Tf saturation, in thalassemia-associated mice (Th3 / +), 6-week-old Th3 / + mice were administered 1 mg / kg LNP-TMPRSS6 siRNA-1 (AD-46273), LNP-Luc control, or PBS via tail vein injection and sacrificed 2 weeks after administration. Five mice were used per group, and data are presented in Figure 7 as mean values ​​+ / - standard deviation. ** indicates a p-value < 0.01 and *** indicates p-value < 0.001. As shown in Figure 7, silencing of TMPRSS6 in Th3 / + mice resulted in significant decreases in serum iron, UIBC, and Tf saturation compared with the control PBS group.

[0359] Effects of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on reticulocyte and erthyrocyte parameters in thalassemia mice (Th3 / +) To evaluate the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on reticulocyte and erythrocyte parameters, including reticulocyte count, reticulocyte hemoglobin content (CHr), and red blood cell count (RBC) in thalassemia-associated mice (Th3 / +), 6-week-old Th3 / + mice were administered 1 mg / kg LNP-TMPRSS6 siRNA-1 (AD-46273), LNP-Luc control, or PBS via tail vein injection. Mice were sacrificed 2 weeks post-administration. Reticulocyte and erythrocyte parameters, including reticulocyte count, reticulocyte hemoglobin content (CHr), and red blood cell count (RBC), were assayed using an Advia 120 analyzer. Five mice were used per group. Data are presented in Figures 8A-8C as mean values ​​+ / - standard deviation. ** indicates a p-value < 0.01 and ***Indicates a p-value <0.001. As shown in Figures 8A and 8B, respectively, silencing TMPRSS6 in Th3 / + mice resulted in a significant decrease in reticulocyte count and hemoglobin content of reticulocytes (Chr). In addition, silencing TMPRSS6 in Th3 / + mice resulted in a significant increase in mature red blood cell (RBC) count (Figure 8C), demonstrating significant improvement in ineffective hematopoiesis, extramedullary hematopoiesis, and erythropoiesis.

[0360] Effects of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on hematological parameters in thalassemia mice (Th3 / +) To evaluate the effects of TMPRSS6 siRNA-mediated silencing on hematological parameters, including hematocrit (HCT), hemoglobin (HGB), red blood cell distribution width (RDW), and mean corpuscular volume (MCV), in thalassemia-positive (Th3 / +) mice, 6-week-old Th3 / + mice were administered 1 mg / kg LNP-TMPRSS6 siRNA-1 (AD-46273), LNP-Luc control, or PBS via tail vein injection and sacrificed 2 weeks post-injection. Hematological parameters, including hematocrit (HCT), hemoglobin (HGB), red blood cell distribution width (RDW), and mean corpuscular volume (MCV), were assayed using an Advia 120 analyzer. Using five mice per group, data are presented in Figure 9 as mean values ​​+ / - standard deviation. ** indicates a p-value < 0.01 and *** indicates p<0.001. Silencing of TMPRSS6 in Th3 / + mice resulted in a significant increase in HCT (Figure 9A), a significant increase in HGB (Figure 9B), a significant decrease in RDW (Figure 9C), and a significant decrease in MCV (Figure 9D). The data presented in Figure 9 demonstrate normalization of the β-thalassemia phenotype in these hematological parameters after administration of LNP-TMPRSS6 siRNA-1 (AD-46273).

[0361] Effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on peripheral blood morphology in thalassemia mice (Th3 / +) To evaluate the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on peripheral blood morphology in thalassemia-positive (Th3 / +) mice, 6-week-old Th3 / + mice were administered 1 mg / kg of LNP-TMPRSS6 siRNA-1 (AD-46273) or LNP-Luc control via tail vein injection and sacrificed 2 weeks after administration. May-Grunwald / Gimsa staining at 10x magnification showed a significant reduction in polychromatosis in TMPRSS6 siRNA-treated Th3 / + mice compared with controls, representing a reduction in reticulocyte counts and an overall trend toward normalization of mature red blood cell morphology. May-Grunwald / Gimsa staining at 10x magnification also showed minor anisocytosis induced by WT TMPRSS6 siRNA animals compared with WT control animals.

[0362] Effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on spleen structure in thalassemia mice (Th3 / +) To evaluate the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on splenic structure in thalassemia-positive (Th3 / +) mice, 6-week-old Th3 / + mice were administered 1 mg / kg LNP-TMPRSS6 siRNA-1 (AD-46273), LNP-Luc control, or PBS via tail vein injection and sacrificed two weeks after administration. Hematoxylin and eosin (H&E) staining at 10x magnification showed that compared with controls, Th3 / + mice treated with TMPRSS6 siRNA had normalized splenic structure, including a reduction in sinusoidal extramedullary hematopoiesis and the reappearance of white pulp nodules.

[0363] Effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on spleen and liver iron content in thalassemia mice (Th3 / +) To evaluate the effect of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on spleen and liver iron content in thalassemia mice (Th3 / +), 6-week-old Th3 / + mice were injected with 1 mg / kg LNP-TMPRSS6 via tail vein injection. Mice were administered siRNA-1 (AD-46273), LNP-Luc control, or PBS and sacrificed two weeks after administration. Five mice were used per group. Data are presented in Figures 10A-10C as mean values ​​+ / - standard deviation. ** indicates a p-value < 0.01 and *** Indicates p<0.001. Silencing of TMPRSS6 in Th3 / + mice resulted in a significant decrease in splenic iron content and spleen weight (Figures 10A and 10B, respectively), suggesting normalization of extramedullary hematopoiesis. A trend toward decreased liver iron content was also observed, although this was not statistically significant (Figure 10C).

[0364] The above results demonstrate that silencing TMPRSS6 by systemic administration of formulated siRNA increases HAMP expression to levels sufficient to ameliorate the phenotype in a mouse model of β-thalassemia intermedia. Thus, LNP-TMPRSS6-siRNA is being developed for congenital iron overload disorders (e.g., β-thalassemia intermedia and hereditary hemochromatosis) characterized by abnormally low hepcidin levels.

[0365] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. (Addendum) As a preferred embodiment, the technical concept that can be grasped from the above embodiment will be described. (Item 1) A double-stranded ribonucleic acid (dsRNA) for inhibiting TMPRSS6 expression, A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity to a TMPRSS6 transcript comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in Tables 2, 3, or 4. (Item 2) 2. The dsRNA of item 1, wherein the dsRNA comprises at least one modified nucleotide. (Item 3) 3. The dsRNA of item 2, wherein at least one of the modified nucleotides is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. (Item 4) 3. The dsRNA of item 2, wherein the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base-containing nucleotides. (Item 5) 2. The dsRNA of item 1, wherein the complementary region is at least 17 nucleotides in length. (Item 6) 2. The dsRNA according to item 1, wherein the complementary region is 19 to 21 nucleotides in length. (Item 7) 2. The dsRNA of item 1, wherein the complementary region is 19 nucleotides in length. (Item 8) 2. The dsRNA of item 1, wherein each strand is 30 nucleotides or less in length. (Item 9) 2. The dsRNA of item 1, wherein at least one strand comprises a 3' overhang of at least one nucleotide. (Item 10) 2. The dsRNA of item 1, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides. (Item 11) 2. The dsRNA of item 1, further comprising a ligand. (Item 12) 12. The dsRNA of item 11, wherein the ligand is bound to the 3' end of the sense strand of the dsRNA. (Item 13) 2. The dsRNA of item 1, wherein the complementary region consists of one of the antisense sequences of Table 2, 3 or 4. (Item 14) 2. The dsRNA of item 1, wherein the dsRNA comprises a sense strand consisting of a sense strand sequence selected from Table 2, 3, or 4, and an antisense strand consisting of an antisense sequence selected from Table 2, 3, or 4. (Item 15) A cell containing the dsRNA according to item 1. (Item 16) A pharmaceutical composition for inhibiting the expression of the TMPRSS6 gene, comprising the dsRNA of item 1. (Item 17) 17. The pharmaceutical composition according to item 16, further comprising a lipid formulation. (Item 18) 18. The pharmaceutical composition of item 17, wherein the lipid formulation is a SNALP or XTC formulation. (Item 19) (a) introducing the dsRNA according to item 1 into a cell; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the TMPRSS6 gene, thereby inhibiting expression of the TMPRSS6 gene in the cells; A method for inhibiting TMPRSS6 expression in a cell, comprising: (Item 20) 20. The method of item 19, wherein the expression of TMPRSS6 is inhibited by at least 30%. (Item 21) A method for treating a disorder mediated by TMPRSS6 expression, comprising administering a therapeutically effective amount of the dsRNA described in Item 1 or the pharmaceutical composition described in any one of Items 16 to 18 to a human in need of such treatment. (Item 22) 22. The method of claim 21, wherein the human has a disorder associated with hemochromatosis. (Item 23) 22. The method of claim 21, wherein the human has β-thalassemia. (Item 24) 22. The method of claim 21, wherein the human has beta-thalassemia intermedia. (Item 25) 24. The method of claim 23, wherein administering the dsRNA to the subject causes at least a 10% decrease in serum iron in the subject. (Item 26) 22. The method of item 21, wherein the dsRNA is administered at a concentration of 0.01 mg / kg to 5 mg / kg of subject body weight. (Item 27) A vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity with at least a portion of an mRNA encoding TMPRSS6, the dsRNA is 30 base pairs or less in length, and the dsRNA targets the mRNA for cleavage. (Item 28) 28. The vector of item 27, wherein the complementary region is at least 15 nucleotides in length. (Item 29) 28. The vector according to item 27, wherein the complementary region is 19 to 21 nucleotides in length. (Item 30) A cell containing the vector according to item 27. (Item 31) 2. The dsRNA of item 1, comprising a sense strand consisting of a sequence selected from the group consisting of SEQ ID NO:111, SEQ ID NO:455, SEQ ID NO:109, SEQ ID NO:524, SEQ ID NO:89, SEQ ID NO:494, SEQ ID NO:445, SEQ ID NO:592, SEQ ID NO:47, and SEQ ID NO:540; and an antisense strand consisting of a sequence selected from the group consisting of SEQ ID NO:112, SEQ ID NO:456, SEQ ID NO:110, SEQ ID NO:525, SEQ ID NO:90, SEQ ID NO:495, SEQ ID NO:446, SEQ ID NO:593, SEQ ID NO:48, and SEQ ID NO:541.

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting TMPRSS6 expression, comprising: A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity to a TMPRSS6 transcript comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in Tables 2, 3, or 4.

2. The dsRNA of claim 1, wherein the dsRNA comprises at least one modified nucleotide.

3. 3. The dsRNA of claim 2, wherein at least one of the modified nucleotides is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

4. The dsRNA of claim 2, wherein the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base-containing nucleotides.

5. 2. The dsRNA of claim 1, wherein the complementary region is at least 17 nucleotides in length.

6. 2. The dsRNA of claim 1, wherein the complementary region is 19 to 21 nucleotides in length.

7. 2. The dsRNA of claim 1, wherein the complementary region is 19 nucleotides in length.

8. 2. The dsRNA of claim 1, wherein each strand is 30 nucleotides or less in length.

9. 10. The dsRNA of claim 1, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

10. 10. The dsRNA of claim 1, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

11. The dsRNA of claim 1, further comprising a ligand.

12. 12. The dsRNA of claim 11, wherein the ligand is attached to the 3' end of the sense strand of the dsRNA.

13. 2. The dsRNA of claim 1, wherein the complementary region consists of one of the antisense sequences of Tables 2, 3, or 4.

14. 2. The dsRNA of claim 1, wherein the dsRNA comprises a sense strand consisting of a sense strand sequence selected from Table 2, 3, or 4, and an antisense strand consisting of an antisense sequence selected from Table 2, 3, or 4.

15. A cell containing the dsRNA of claim 1.

16. A pharmaceutical composition for inhibiting the expression of the TMPRSS6 gene, comprising the dsRNA of claim 1.

17. 17. The pharmaceutical composition of claim 16, further comprising a lipid formulation.

18. 18. The pharmaceutical composition of claim 17, wherein the lipid formulation is SNALP or an XTC formulation.

19. (a) introducing the dsRNA of claim 1 into a cell; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcripts of the TMPRSS6 gene, thereby inhibiting expression of the TMPRSS6 gene in the cells; A method for inhibiting TMPRSS6 expression in a cell, comprising:

20. 20. The method of claim 19, wherein the expression of TMPRSS6 is inhibited by at least 30%.

21. A method for treating a disorder mediated by TMPRSS6 expression, comprising administering a therapeutically effective amount of the dsRNA of claim 1 or the pharmaceutical composition of any one of claims 16 to 18 to a human in need of such treatment.

22. 22. The method of claim 21, wherein the human has a disorder associated with hemochromatosis.

23. 22. The method of claim 21, wherein the human has beta thalassemia.

24. 22. The method of claim 21, wherein the human has beta-thalassemia intermedia.

25. 24. The method of claim 23, wherein administering the dsRNA to the subject causes at least a 10% decrease in serum iron in the subject.

26. 22. The method of claim 21, wherein the dsRNA is administered at a concentration of 0.01 mg / kg to 5 mg / kg of the subject's body weight.

27. A vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity with at least a portion of an mRNA encoding TMPRSS6, the dsRNA is 30 base pairs or less in length, and the dsRNA targets the mRNA for cleavage.

28. 28. The vector of claim 27, wherein the region of complementarity is at least 15 nucleotides in length.

29. 28. The vector of claim 27, wherein the complementary region is 19 to 21 nucleotides in length.

30. A cell comprising the vector of claim 27.

31. 2. The dsRNA of claim 1, comprising a sense strand consisting of a sequence selected from the group consisting of SEQ ID NO:111, SEQ ID NO:455, SEQ ID NO:109, SEQ ID NO:524, SEQ ID NO:89, SEQ ID NO:494, SEQ ID NO:445, SEQ ID NO:592, SEQ ID NO:47, and SEQ ID NO:540; and an antisense strand consisting of a sequence selected from the group consisting of SEQ ID NO:112, SEQ ID NO:456, SEQ ID NO:110, SEQ ID NO:525, SEQ ID NO:90, SEQ ID NO:495, SEQ ID NO:446, SEQ ID NO:593, SEQ ID NO:48, and SEQ ID NO:541.

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