(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/051135 A2 9 April 2015 (09.04.2015)

(51) International Patent Classification: [US/US]; Novartis Institutes for BioMedical Research, C12N 15/113 (2010.01) Inc., 250 Massachusetts Avenue, Cambridge, Massachu setts 02139 (US). GOSSERT, Alvar [CH/CH]; Novartis (21) Number: International Application Pharma AG, Lichtstrasse 35, CH-4056 Basel (CH). PCT/US20 14/05885 1 GREENIDGE, Paulette [GB/CH]; Novartis Pharma AG, (22) International Filing Date: Lichtstrasse 35, CH-4056 Basel (CH). HUESKEN, Dieter 2 October 2014 (02. 10.2014) [DE/CH]; Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel (CH). HUNZIKER, Juerg [CH/CH]; Novartis (25) Filing Language: English Pharma AG, Lichtstrasse 35, CH-4056 Basel (CH). NATT, (26) Publication Language: English Francois Jean-Charles [FR/CH]; Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel (CH). PATNAIK, Anup (30) Priority Data: [IN/US]; Novartis Institutes for BioMedical Research, Inc., 61/886,753 4 October 201 3 (04. 10.2013) US 250 Massachusetts Avenue, Cambridge, Massachusetts 61/930,681 23 January 2014 (23.01.2014) US 02139 (US). PATTERSON, Andrew [US/US]; Novartis (71) Applicant (for all designated States except US) : NO- Institutes for BioMedical Research, Inc., 250 Massachu VARTIS AG [CH/CH]; Lichtstrasse 35, CH-4056 Basel setts Avenue, Cambridge, Massachusetts 02139 (US). (CH). RONDEAU, Jean-Michel Rene [FR/CH]; Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel (CH). SO¬ (72) Inventors; and LOMON, Jonathan [US/US]; Novartis Institutes for Bio (71) Applicants (for US only): BARYZA, Jeremy Lee Medical Research, Inc., 250 Massachusetts Avenue, Cam [US/US]; Novartis Institutes for BioMedical Research, bridge, Massachusetts 02139 (US). WEILER, Jan Inc., 250 Massachusetts Avenue, Cambridge, Massachu [DE/US]; Novartis Institutes for BioMedical Research, setts 02139 (US). BLOMMERS, Marcel [NL/CH]; N o Inc., 250 Massachusetts Avenue, Cambridge, Massachu vartis Pharma AG, Lichtstrasse 35, CH-4056 Basel (CH). setts 021 39 (US). ZHU, Meicheng [US/US]; Novartis In BORLAND, Todd [US/US]; Novartis Institutes for Bio- stitutes for BioMedical Research, Inc., 250 Massachusetts Medical Research, Inc., 250 Massachusetts Avenue, Cam Avenue, Cambridge, Massachusetts 02139 (US). bridge, Massachusetts 02139 (US). FERNANDEZ, Cesar [CH/CH]; Novartis Pharma AG, Lichtstrasse 35, CH-4056 (74) Common Representative: NOVARTIS AG; Lichtstrasse Basel (CH). GEBUHR, Tom [US/US]; Novartis Institutes 35, CH-4056 Basel (CH). for BioMedical Research, Inc., 250 Massachusetts Avenue, (81) Designated States (unless otherwise indicated, for every Cambridge, Massachusetts 02139 (US). GENO, Erin kind of national protection available): AE, AG, AL, AM,

[Continued on nextpage]

(54) Title: ORGANIC COMPOSITIONS TO TREAT HEPCIDIN-RELATED DISEASES

FIG. 1

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© (57) Abstract: The present disclosure relates to methods of treating Hepcidin-related diseases such as anemia, iron-deficient eryth- o ropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (AI) (sometimes designated an emia of chronic disease or ACD), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimers disease, Parkinsons disease and Friedrichs ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and hair loss, using a therapeutically effective amount of a RNAi agent to Hepcidin. w o 2015/051135 A2 III! II II 11III III UN I II III I I III II I II

AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, Published: VN, ZA, ZM, ZW. — without international search report and to be republished upon receipt of that report (Rule 48.2(g)) (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, ORGANIC COMPOSITIONS TO TREAT HEPCIDIN-RELATED DISEASES

[001] FIELD OF THE INVENTION [002] The present disclosure pertains to RNAi agents to Hepcidin (HAMP)

[003] In various embodiments, the disclosure pertains to a Hepcidin RNAi agent comprising a first and a second strand, wherein the sequence of the first and/or second strand is that of any sequence disclosed herein.

[004] In various embodiments, the disclosure pertains to Hepcidin RNAi agents which comprise any sequence or an 18-nt portion of any Hepcidin RNAi agent sequence disclosed herein (e.g., nt 1-18 or nt 2-19 of any sequence disclosed herein). In various embodiments, these Hepcidin RNAi agents are blunt-ended.

[005] In various embodiments, the disclosure pertains to Hepcidin RNAi agents which comprise any sequence of at least 15 contiguous nt of any Hepcidin RNAi agent sequence disclosed herein (e.g., nt 1-15, 2-16, 3-17, 4-18, 5-19, etc., of any sequence disclosed herein).

[006] In various embodiments, the disclosure relates to compositions comprising a Hepcidin RNAi agent (an RNAi agent targeting Hepcidin) having a novel format (the "18-mer format"). These Hepcidin RNAi agents comprise a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the 3' end of both the sense and anti-sense strand terminate in a phosphate or modified internucleoside linker and further comprise, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the disclosure pertains to a Hepcidin RNAi agent that comprises a first and a second strand, wherein the first and second strand are both 18-mers, and the first and second strand together form a blunt-ended duplex, and wherein the 3' end of the first strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer, a phosphate or modified internucleoside linker, and a 3' end cap; and wherein the 3' end of the second strand terminates in a phosphate or modified internucleoside linker and further comprises a 3' end cap. In some embodiments, the disclosure pertains to a Hepcidin RNAi agent that comprises a first and a second strand, wherein the first and second strands are both 18-mers, and the first and second strand together form a blunt-ended duplex, and wherein the 3' end of both the first and second strand terminate in a phosphate or modified internucleoside linker and both further comprise a 3' end cap. In some embodiments, the first strand is the antisense strand and the second strand is the sense strand. In other embodiments, the first strand is the sense strand and the second strand is the antisense strand. The two strands can have the same or different spacers, phosphates or modified internucleoside linkers, and/or 3' end caps. The strands can be ribonucleotides, or, optionally, one or more nucleotide can be modified or substituted. Optionally, at least one nucleotide comprises a modified internucleoside linker. Optionally, the RNAi agent can be modified on one or both 5' end. Optionally, the sense strand can comprise a 5' end cap which reduces the amount of the RNA interference mediated by this strand. Optionally, the RNAi agent is attached to a ligand. The disclosure also relates to processes for making such compositions, and methods and uses of such compositions, e.g., to mediate RNA interference.

[007] BACKGROUND OF THE INVENTION [008] Control of iron absorption, storage and transport are necessary to prevent iron deficiency and avoid oxidative damage due to free iron. Loss of this control is implicated in various diseases, such as anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia. Ganz et al. 2003 Blood 102: 783-788; Yuan et al. 2003 Ann. Med. 35: 578-591 ; O'Neil et al. 2005 Sem. Liver Dis. 25: 381-391; and Moos et al. 2004 Ann. New York Acad. Sci. 1012: 14- 26. Genetic and clinic observational studies have highlighted that aberrant iron localization in cells or tissues may be more important than overall body iron content in iron-related disease. Mackenzie et al. 2008 Antiox. Redox Sign. 10: 997-1030; and Cartwright 1966 Sem. Hemat. 3: 351-375. Despite this, therapeutic control of iron metabolism has only involved manipulation of total body iron levels either by iron administration, chelation therapy or phlebotomy. Targeting control of iron distribution in the body may therefore represent an attractive therapeutic approach to treat disease.

[009] There thus exists the need for additional therapies for treatment of anemia and related diseases.

[0010] BRIEF SUMMARY OF THE INVENTION [0011] The present disclosure encompasses RNAi agents to Hepcidin, for inhibition of Hepcidin, and which are useful in treatment of a Hepcidin-related disease, such as anemia. [0012] The present disclosure also encompasses a method for inhibiting and/or decreasing the expression and/or activity of Hepcidin in a human subject. In various embodiments, the subject has a Hepcidin-related disease (a pathological state mediated at least in part by Hepcidin over-expression, over-abundance or hyper-activity), and the method comprises the step of administering to the subject a therapeutically effective amount of a RNAi agent to

Hepcidin. In various embodiments, the Hepcidin-related disease is selected from: anemia, iron- deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al) (sometimes designated anemia of chronic disease or ACD), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and hair loss. In some embodiments, the subject is a dialysis patient. In some embodiments, the Hepcidin-related disease is anemia and the subject is a dialysis patient.

[0013] In various embodiments, the present disclosure pertains to a RNAi agent to Hepcidin, comprising two strands, wherein the sequence of the first and/or second strand is, comprises, comprises 15 contiguous nt of, or comprises a sequence of 15 contiguous nt with 0- 3 mismatches from: the sequence of any RNAi agent disclosed herein.

[0014] In some embodiments, the RNAi agent to Hepcidin comprises a first strand and a second strand, wherein the sequence of the first strand is, comprises, comprises 15 contiguous nt of, or comprises 15 contiguous nt of (with 1-3 mismatches from) any sequence of any Hepcidin RNAi agent listed herein. [0015] The present disclosure provides specific RNAi agents for inhibition of Hepcidin, and methods that are useful in reducing Hepcidin levels in a subject, e.g., a mammal, such as a human. The present disclosure specifically provides double-stranded Hepcidin RNAi agents comprising at least 15, 16, 17, 18, or 19 or more contiguous nucleotides of Hepcidin. In particular, the present disclosure provides Hepcidin RNAi agents comprising sequences of 15 or more contiguous nucleotides differing by 0, 1, 2 or 3 from those of any of the RNAi agents provided, e.g., in any table herein, such as Tables 1 to 4 and 6. The RNAi agents particularly can in one aspect comprise less than 30 nucleotides per strand, e.g., such as 17-23 nucleotides, 15-19, 18-22, and/or 19-21 nucleotides, and/or such as those provided, e.g., in

Tables 1 to 4 and 6, and modified and unmodified variants thereof (e.g., wherein the sense and/or anti-sense or first and/or second strand are modified or unmodified). The present disclosure also provides Hepcidin RNAi agents comprising a sense strand and an anti-sense strand, wherein the sense and/or the anti-sense strand comprise sequences of 19 or more contiguous nucleotides differing by 0, 1, 2 or 3 from those of the RNAi agents provided, e.g., in

Tables 1 to 4 and 6, and modified or unmodified variants thereof. The sense and anti-sense strand can be contiguous, or physically connected, e.g., by covalently bonds, a loop or linker. [0016] The double-stranded Hepcidin RNAi agents can have 0, 1 or 2 blunt ends, and/or overhangs of 1, 2, 3 or 4 nucleotides (i.e., 1 to 4 nt) from one or both 3' and/or 5' ends. The double-stranded Hepcidin RNAi agents can also optionally comprise one or two 3' caps and/or one or more modified nucleotides. Modified variants of sequences as provided herein include those that are otherwise identical but contain substitutions of a naturally-occurring nucleotide for a corresponding modified nucleotide. [0017] Furthermore, the Hepcidin RNAi agent can either contain only nucleotides, e.g., naturally-occurring ribonucleotide subunits, or optionally one or more modifications to the sugar, phosphate or base of one or more of the substitute nucleotide subunits, whether they comprise ribonucleotide subunits or deoxyribonucleotide subunits. In one aspect, modified variants of the disclosed RNAi agents have a thymidine (as RNA, or, preferably, DNA) replacing a uridine, or have an inosine base. In some aspects, the modified variants of the disclosed RNAi agents can have a nick in the passenger strand, mismatches between the guide and passenger strand, DNA replacing the RNA of a portion of both the guide and passenger strand (e.g., the seed region), and/or a shortened passenger strand (e.g., 15, 16, 17 or 18 nt). Once a functional guide strand is identified, modifications and variants of the RNAi agent can be readily made. Any two or more modifications which are not mutually exclusive can be combined (e.g., the combination of base modifications with shortened passenger strand; or nicked passenger strand and base modifications; or DNA replacing part or all of the seed region and base modifications in the remaining RNA; etc.).

[0018] In one aspect, modified variants of the disclosed RNAi agents include RNAi agents with the same sequence (e.g., the same sequence of bases) as any RNAi agent disclosed in any of Tables 1 to 4 and 6 . It is also noted that various authors have shown that once a successful RNAi agent is designed, generally adding a few nt to one or both strands does not impair RNA activity. Thus, this disclosure encompasses RNAi agent comprising a first strand and a second strand, wherein the sequence of the first and/or second strand comprises the sequence of any Hepcidin RNAi agent disclosed herein, further comprising 1-5 nt. [0019] In one aspect, modified variants of the disclosed Hepcidin RNAi agents include RNAi agents with the same sequence (e.g., the same sequence of bases) as any RNAi agent disclosed in any of Tables 1 to 4 and 6, but with one or more modifications to one or more of the sugar or phosphate of one or more of the nucleotide subunits. In one aspect, the modifications improve efficacy, stability (e.g., against nucleases in, for example, blood serum or intestinal fluid), and/or reduce immunogenicity of the RNAi agent. One aspect of the present disclosure relates to a double-stranded oligonucleotide comprising at least one non-natural nucleobase. In certain aspects, the non-natural nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a particular aspect, the non-natural nucleobase is difluorotolyl. In certain aspects, only one of the two oligonucleotide strands contains a non-natural nucleobase. In certain aspects, both of the oligonucleotide strands contain a non-natural nucleobase.

[0020] In various embodiments, the disclosure relates to compositions comprising a Hepcidin RNAi agent having a novel format (the "18-mer format"). These Hepcidin RNAi agents comprise a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the 3' end of both the sense and anti-sense strand terminate in a phosphate or modified internucleoside linker and further comprise, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the disclosure pertains to a Hepcidin RNAi agent that comprises a first and a second strand, wherein the first and second strand are both 18-mers, and the first and second strand together form a blunt- ended duplex, and wherein the 3' end of the first strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer, a phosphate or modified internucleoside linker, and a 3' end cap; and wherein the 3' end of the second strand terminates in a phosphate or modified internucleoside linker and further comprises a 3' end cap. In some embodiments, the disclosure pertains to a Hepcidin RNAi agent that comprises a first and a second strand, wherein the first and second strands are both 18-mers, and the first and second strand together form a blunt-ended duplex, and wherein the 3' end of both the first and second strand terminate in a phosphate or modified internucleoside linker and both further comprise a 3' end cap. In some embodiments, the first strand is the antisense strand and the second strand is the sense strand. In other embodiments, the first strand is the sense strand and the second strand is the antisense strand. The two strands can have the same or different spacers, phosphates or modified internucleoside linkers, and/or 3' end caps. The strands can be ribonucleotides, or, optionally, one or more nucleotide can be modified or substituted. Optionally, at least one nucleotide comprises a modified internucleoside linker. Optionally, the RNAi agent can be modified on one or both 5' end. Optionally, the sense strand can comprise a 5' end cap which reduces the amount of the RNA interference mediated by this strand. Optionally, the RNAi agent is attached to a ligand. The disclosure also relates to processes for making such compositions, and methods and uses of such compositions, e.g., to mediate RNA interference. [0021] The Hepcidin RNAi agent(s) can optionally be attached to a ligand selected to improve one or more characteristic, such as, e.g., stability, distribution and/or cellular uptake of the agent, e.g., cholesterol or a derivative thereof. The RNAi agent(s) can be isolated or be part of a pharmaceutical composition used for the methods described herein. Particularly, the pharmaceutical composition can be formulated for delivery to specific tissues (e.g., those afflicted with a Hepcidin-related disease) or formulated for parenteral administration. The pharmaceutical composition can optionally comprise two or more RNAi agents, each one directed to the same, overlapping or a different segment of the Hepcidin mRNA. Optionally, the pharmaceutical composition can further comprise or be used in conjunction with any known treatment for any Hepcidin-related disease. [0022] Second agent or treatment. The method also optionally further comprises the step of administering a second agent or treatment. In some aspects, this second agent is another

RNAi agent to Hepcidin. In various embodiments, the second agent or treatment is iron administration, chelation therapy, phlebotomy, erythropoiesis stimulating agent (ESA) (e.g.,

Epoetin alfa or darbepoetin alfa), anti-Hepcidin antibody, hemodialysis, or hyperbaric oxygen. In other aspects, the second agent or treatment is directed to another target, which is also hyper active, mutated and/or over-expressed in the pathological state. [0023] Methods. The present disclosure further provides methods for reducing the level of Hepcidin mRNA in a cell, particularly in the case of a disease characterized by over-expression, over-abundance or hyper-activity of Hepcidin. Cells comprising an alteration such as a mutation, over-expression and/or hyperactivity of Hepcidin are termed "Hepcidin-defective" cells. Such methods comprise the step of administering one or more of the RNAi agents of the present disclosure to an Hepcidin-defective cell, as further described below. The present methods utilize the cellular mechanisms involved in RNA interference to selectively degrade the target RNA in a cell and are comprised of the step of contacting a cell with one of the RNAi agents of the present disclosure. [0024] The present disclosure also encompasses a method of treating a human subject having a pathological state mediated at least in part by Hepcidin over-expression, over abundance or hyper-activity, the method comprising the step of administering to the subject a therapeutically effective amount of a RNAi agent Hepcidin. Additional methods involve preventing, treating, modulating and/or ameliorating a pathological state wherein disease progression requires Hepcidin, although Hepcidin is not amplified or over-expressed. Such methods comprise the step of administering one of the RNAi agents of the present disclosure to a subject, as further described below. Such methods can be performed directly on a cell or can be performed on a mammalian subject by administering to a subject one of the RNAi agents/pharmaceutical compositions of the present disclosure. Reduction of target Hepcidin mRNA in a cell results in a reduction in the amount of encoded Hepcidin protein produced. In an organism, this can result in restoration of balance in a pathway involving Hepcidin, and/or prevention of Hepcidin accumulation, and/or a reduction in Hepcidin activity and/or expression, and/or prevention of Hepcidin-mediated activation of other genes, and/or amelioration, treatment and/or prevention of a Hepcidin-related disease. In one aspect, a reduction in Hepcidin expression, level or activity can limit disease growth. [0025] The methods and compositions of the present disclosure, e.g., the methods and Hepcidin RNAi agent compositions, can be used in any appropriate dosage and/or formulation described herein or known in the art, as well as with any suitable route of administration described herein or known in the art. [0026] The details of one or more aspects of the present disclosure are set forth in the accompanying drawings and the description below. Elements of the various aspects (e.g., sequences, modifications, substitutions, spacers, modified internucleoside linkers, endcaps, combinations of RNAi agents, delivery vehicles, combination therapy involving a Hepcidin RNAi agent and another agent, etc.) disclosed herein or known in the art which are not mutually exclusive can be combined with each other, provided that the agent or agents are still capable of mediating RNA interference. For example, any RNAi agent sequence disclosed herein can be combined with any set of modifications or endcaps disclosed herein. Similarly, any combination of modifications, 5' end caps, and/or 3' end caps can be used with any RNAi agent sequence disclosed herein. Any RNAi agent disclosed herein (with any combination of modifications or endcaps or without either modifications or endcaps) can be combined with any other RNAi agent or other treatment composition or method disclosed herein. [0027] Other features, objects, and advantages of the present disclosure will be apparent from this description, the drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0028] FIG. 1 shows the efficacy of various Hepcidin RNAi agents in vitro. [0029] FIGs. 2A and 2B show residual expression level, indicating in vitro RNA interference or KD (knockdown) mediated by various Hepcidin RNAi agents comprising a 3' end cap: BP (biphenyl), C6, X027, X038, X050, X051 , X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068, and X069 on the guide strand, as described in Example 3A. These RNAi agents are without a 2'-MOE clamp (-) or with a 2'-MOE clamp (MOE); or without a ribitol spacer (-) or with a ribitol spacer (rib). Descriptions for FIG. 2A are provided at the bottom of FIG 2B.

[0030] FIGs. 3A and 3B detail some of the Hepcidin RNAi agents used in the data shown in FIGS 2A and 2B and others. The sequences in FIG. 3A are represented by, from top to bottom, by SEQ ID NOs: 172 to 177 (400), 178 to 183 (402) and 184 (400 21-mer). The sequences in

FIG. 3B are represented, from top to bottom, by SEQ ID NOs: 185 to 190 (400); 191 to 196 (402) and 197 (400 21-mer). [0031] FIG. 3C represents modified variants of Hepcidin RNAi agents, wherein some RNA nucleotides are substituted with DNA, and some RNA nucleotides are modified as 2'-OMe or 2'- MOE. The sequences are, from top to bottom, SEQ ID NOs: 198 to 203 (400) and 204 to 209 (402). The DNA is well-tolerated in the seed region of the RNAi agent and can reduce the HeLa phenotype (i.e., reduces off-target effects as measured by toxicity to HeLa cells which do not express Hepcidin). [0032] Fig. 4 (TOP) shows a non-limiting example of the 18-mer RNAi agent format. "L" indicates a non-nucleotidic "Ngand", e.g., any of various 3' end caps which can be used (e.g., PAZ Ligands). This generic sequence is represented by SEQ ID NOs: 210 and 2 11. Fig. 4 (MIDDLE) also shows specific examples of a Hepcidin RNAi agent, comprising at the 3' termini of various strands, in 5' to 3' order, and bound to the 3' terminal phosphate: a ribitol spacer (rib), a phosphate (p) and a 3' end cap (X058 or C6). These sequences are represented by SEQ ID NOs: 212 and 213 (400) and 214 and 215 (402). Various modifications are also shown (BOTTOM). [0033] FIG. 5 shows the structures of example RNAi agents comprising a spacer [which is a ribitol (rib), C3 or 4-methoxybutane-1,3-diol (A5300)]; a phosphate; and a 3' end cap which is X058. The Figure depicts the spacers in the context of an 18-mer RNAi agent and a specific 3' end cap, but the spacers can be used with any RNAi agent strand of any length, sequence or target, and with any 3' end cap. [0034] FIG. 6 shows a comparison of corresponding 19-mer and 18-mer format human Hepcidin RNAi agents. RNAi agents include: hsJHAMP (human HAMP) 36, 37, 93, 160, 185, 232, 296, 299, 300, 301, 309, 312, 325, 328, 332, 397, 398, 400, 401 , 402, 403 (starting position).

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure encompasses RNAi agents to Hepcidin, for targeting and inhibition of Hepcidin, which are useful in treatment of Hepcidin-related diseases (e.g., diseases associated with mutations in and/or altered expression, level and/or activity of Hepcidin, diseases requiring Hepcidin, diseases affected by a factor whose expression, over-expression, or hyper-activity is directly or indirectly affected by Hepcidin, and/or diseases treatable by modulating the expression, level and/or activity of Hepcidin). Such Hepcidin-related diseases include those listed herein or known in the art. The present disclosure also provides methods of treating a human subject having a pathological state mediated at least in part by Hepcidin over- expression, over-abundance or hyper-activity, or requiring Hepcidin, the method comprising the step of administering to the subject a therapeutically effective amount of a RNAi agent to Hepcidin.

[0036] Various aspects of the disclosure include the following: [0037] An RNAi agent comprising an antisense strand of an RNAi agent described herein.

[0038] In one aspect, an aspect of the present disclosure relates to a composition comprising a Hepcidin RNAi agent comprising an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides (nt) differing by 0, 1, 2 , or 3 nucleotides from the antisense strand of an RNAi agent to Hepcidin selected from any sequence provided herein (e.g., in any one or more of Tables 1 to 4 and 6, etc.). In another aspect, the present disclosure relates to a composition comprising a Hepcidin RNAi agent comprising a first strand and a second strand, wherein the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the first strand of an RNAi agent to Hepcidin from any sequence provided herein. In another aspect, the present disclosure relates to a composition comprising a Hepcidin RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nucleotides from the sense strand and the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nucleotides from the antisense strand of an RNAi agent to

Hepcidin listed immediately above. In some embodiments, one or both strands of the Hepcidin RNAi agent is an 18-mer. In one embodiment, both strands are 18-mers and together they form a blunt-ended duplex. In another aspect, the present disclosure relates to a composition comprising a Hepcidin RNAi agent comprising a first strand and a second strand, wherein the sequence of the first strand comprises the sequence of the first strand of any sequence provided herein. In another aspect, the present disclosure relates to a composition comprising a Hepcidin RNAi agent comprising a first strand and a second strand, wherein the sequence of the first strand is the sequence of the first strand of any sequence provided herein. In various aspects, the first and second strands are the anti-sense and sense strand, respectively, of any

RNAi agent disclosed herein. In various aspects, the first and second strands are the sense and anti-sense strand, respectively, of any RNAi agent disclosed herein.

[0039] In various embodiments, the disclosure relates to compositions comprising a Hepcidin RNAi agent having a novel format (the "18-mer format"). These Hepcidin RNAi agents comprise a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the 3' end of both the sense and anti-sense strand terminate in a phosphate or modified internucleoside linker and further comprise, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the disclosure pertains to a Hepcidin RNAi agent that comprises a first and a second strand, wherein the first and second strand are both 18-mers, and the first and second strand together form a blunt- ended duplex, and wherein the 3' end of the first strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer, a phosphate or modified internucleoside linker, and a 3' end cap; and wherein the 3' end of the second strand terminates in a phosphate or modified internucleoside linker and further comprises a 3' end cap. In some embodiments, the disclosure pertains to a Hepcidin RNAi agent that comprises a first and a second strand, wherein the first and second strands are both 18-mers, and the first and second strand together form a blunt-ended duplex, and wherein the 3' end of both the first and second strand terminate in a phosphate or modified internucleoside linker and both further comprise a 3' end cap. In some embodiments, the first strand is the antisense strand and the second strand is the sense strand. In other embodiments, the first strand is the sense strand and the second strand is the antisense strand. The two strands can have the same or different spacers, phosphates or modified internucleoside linkers, and/or 3' end caps. The strands can be ribonucleotides, or, optionally, one or more nucleotide can be modified or substituted. Optionally, at least one nucleotide comprises a modified internucleoside linker. Optionally, the RNAi agent can be modified on one or both 5' end. Optionally, the sense strand can comprise a 5' end cap which reduces the amount of the RNA interference mediated by this strand. Optionally, the RNAi agent is attached to a ligand. The disclosure also relates to processes for making such compositions, and methods and uses of such compositions, e.g., to mediate RNA interference. In some embodiments, the disclosure pertains to an RNAi agent comprising any sequence disclosed herein. In some embodiments, the disclosure pertains to an RNAi agent comprising a first strand and a second strand, wherein the sequence of the first or second strand is the sequence of any RNAi agent strand disclosed herein. [0040] Particular duplexes include the unmodified (e.g., "generic") and example modified variants listed in Tables 1 to 4 and 6 and otherwise disclosed herein; additional sequences and data for these RNAi agents are presented in the subsequent Tables. In addition to the described example modifications, other modified variants can be made using the nucleotide sequences provided.

[0041] TABLES TABLE 1. 19-MER TARGET SEQUENCES (DNA) TABLE 2 . 19-MER TARGET SEQUENCES (RNA) TABLE 3. TOP 6 CANDIDATES. 18-mer SEQUENCES TABLE 4 . HEPCIDIN RNAi AGENTS 400 and 402 IN VARIOUS FORMATS

[0042] In various aspects, the first and/or second strand are 18- or 19-mers, e.g., as presented in Tables 1 to 4 . [0043] The names in these Tables are sometimes prefixed with "Hepcidin" or "Hepcidin (e.g., "400" is also known as "Hepcidin 400" or Hepcidin_400"). For some hepcidin RNAi agents, the prefix "hsJHAMP" is used, indicating human (hs, Homo sapiens) HAMP (Hepcidin anti-microbial protein or peptide). Thus, "400" is also "hs_HAMP_400". Thus, "400", "hs_HAMP_400", and "Hepcidin_400" and the like all indicate RNAi agents of the same sequence, although they may differ in modifications. For some of these sequences, they may also differ in length (18-mer, 19-mer, 21-mer, etc.) though they all have the same starting position. [0044] For other agents, "mmJHAMP" is used, indicating a sequence of mouse (Mus musculus) origin. [0045] One or both strands of the 18-mer format can comprise, in various embodiments, in 5' to 3' order, a 18-mer, a spacer, and a 3' end cap. The 18-mer can be modified or unmodified.

In various embodiments, the modifications include the two nt on the 3' end being 2'-MOE (a "2- MOE clamp"). In various embodiments, with or without the 2'-MOE clamp, various nucleotides can be modified with a 2'-MOE, 2'-OMe, 2'-F, or substituted with DNA, a peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), unlocked nucleic acid (UNA). The spacer can be ribitol or other type of abasic nucleotide, 2'-deoxy-ribitol, diribitol, 2'- methoxyethoxy-ribitol (ribitol with 2 -MOE), C3, C4, C5, C6, or 4-methoxybutane-1,3-diol (5300). The 3' end cap can be selected from any of various 3' end caps described herein or known in the art. The efficacy of various human Hepcidin RNAi agents wherein the anti-sense (guide) strand of an 18-mer 400 or 402 sequence is modified are shown in Fig. 2A and 2B. [0046] Various non-limiting examples of modified forms of sequences hs_HAMP_400 and 402 are shown in Figs. 3A to 5 . [0047] In one embodiment, at least one nucleotide of the RNAi agent is modified. [0048] In one embodiment, said at least one modified nucleotide is selected from among 2' alkoxyribonucleotide, 2' alkoxyalkoxy ribonucleotide, or 2'-fluoro ribonucleotide. In another embodiment, said at least one modified nucleotide is selected from 2'-OMe, 2'-MOE and 2'-H. In various aspects, the nucleotide subunit is chemically modified at the 2' position of the sugar. In one aspect, the 2' chemical modification is selected from a halo, a C1-10 alkyl, a C1-10 alkoxy, a halo, and the like. In specific aspects, the 2' chemical modification is a C1-10 alkoxy selected from -OCH 3 (i.e., "OMe"), -OCH 2CH3 (i.e., "OEt") or -CH2OCH2CH3 (i.e., methoxyethyl or "MOE"); or is a halo selected from F. [0049] In various embodiments, one or more nucleotides is modified or is DNA or is replaced by a peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 - fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), and/or unlocked nucleic acid (UNA); and/or at least one nucleotide comprises a modified internucleoside linker (e.g., wherein at least one phosphate of a nucleotide is replaced by a modified internucleoside linker), wherein the modified internucleoside linker is selected from phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker, and a compound of formula (I) (as described elsewhere herein).

[0050] In one embodiment, the first two base-pairing nucleotides on the 3' end of the first and/or second strand are modified.

[0051] In one embodiment, the first two base-pairing nucleotides on the 3' end of the first and/or second strand are 2'-MOE.

[0052] In various sequences disclosed herein, lower-case letters (e.g., c, u) indicate modified nucleotides while upper case letters (e.g., C, U, A , G) indicate unmodified nucleotides.

In the Tables herein, example modified versions of each of the sequences are shown. However, the present disclosure also contemplates and encompasses unmodified versions of these sequences and other versions which comprise additional or alternative modifications.

[0053] In various sequences disclosed herein, the modified and unmodified variants can optionally further comprise the sequence "TT", "dTdT", "dTsdT" or "UU" as an example of a single-stranded overhang at the 3' end, also termed herein a terminal dinucleotide or 3' terminal dinucleotide. dT is 2'-deoxy-thymidine-5'-phosphate and sdT is 2'-deoxy Thymidine 5'- phosphorothioate. In the disclosed sequences, terminal dinucleotide "UU" is UU or 2'-OMe-U 2'-OMe-U, and the terminal TT and the terminal UU can be in the inverted/reverse orientation. The terminal dithymidine (e.g., UU) is not part of the Hepcidin target sequence, but is a modified variant of the dithymidine dinucleotide commonly placed as an overhang to protect the ends of siRNAs from nucleases (see, for example, Elbashir et al. 2001 Nature 4 11: 494-498; Elbashir et al. 2001 EMBO J. 20: 6877-6888; and Kraynack et al. 2006 RNA 12:163-176). A terminal dinucleotide is known from these references to enhance nuclease resistance but not contribute to target recognition. Thus, the present disclosure also encompasses any modified or any unmodified variant disclosed herein, wherein the modified variant comprises a terminal TT, dTdT, sdT, dTsdT, sdTsdT, sdTdT, or the like which may be in either the inverted/reverse orientation or in the same 5' to 3' orientation as the Hepcidin specific sequence in the duplex. In addition, terminology used herein referring to "the Hepcidin portion of a RNAi agent sequence" and the like indicate the portion of the sequence of a RNAi agent which is derived from Hepcidin (thus "the Hepcidin portion of a RNAi agent sequence" does not include, for example, a terminal dTdT, TT, UU, U (2'-OMe) dT, U (2'-OMe) U (2'-OMe), T(2'-OMe) T (2'-OMe), T(2'-OMe) dT, or the like, but does include the portion of the RNAi agent that corresponds to or is complementary to a portion of the Hepcidin gene sequence or mRNA sequence. In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein, further comprising a 3' terminal dinucleotide (a single-stranded overhang comprising 2 nt at the 3' end). In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein, further comprising a 3' terminal dinucleotide selected from TT, UU, U (2'-OMe) dT, U (2'-OMe) U (2'-OMe), T(2'-OMe) T (2'-OMe), T(2'-OMe) dT, dTdT, sdT, dTsdT, sdTsdT, and sdTdT. In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein, further comprising a 3' terminal UU dinucleotide. [0054] On any modified or unmodified variant, a 3' end cap, as is known in the art, can be used instead of or in addition to a terminal dinucleotide to stabilize the end from nuclease degradation provided that the 3' end cap is able to both stablize the RNAi agent (e.g., against nucleases) and not interfere excessively with siRNA activity. In various embodiments, a 3' end cap is used in place of a dinucleotide overhang. Thus, the present disclosure also encompasses any modified or any unmodified variant disclosed herein, wherein the modified variant further comprises a terminal 3' end cap.

[0055] An RNAi agent comprising an antisense strand of an RNAi agent described herein.

[0056] In one particular specific aspect, the present disclosure relates to a composition comprising an RNAi agent comprising an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of an RNAi agent to Hepcidin selected from those antisense strands in the specific duplexes provided herein. [0057] Various particular specific aspects of this aspect are described below. [0058] In one aspect, the composition further comprises a second RNAi agent to Hepcidin.

In various aspects, the second RNAi agent is physically separate from the first, or the two are physically connected (e.g., covalently linked or otherwise conjugated). [0059] In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein. In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein, further comprising an additional about 6 to 20 nucleotides on one or both strands (e.g., about 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nt). In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein, further comprising a 3' terminal dinucleotide. In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein, further comprising a 3' terminal dinucleotide selected from TT, UU, U (2'-OMe) dT, U (2 - OMe) U (2'-OMe), T(2'-OMe) T (2'-OMe), T(2'-OMe) dT, dTdT, sdT, dTsdT, sdTsdT, and sdTdT. In one aspect, the composition comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand and the sequence of the second strand are the sequences of the first and second strand, respectively, of any RNAi agent provided herein, further comprising a 3' terminal UU dinucleotide. In various aspects, the first and second strands are the sense and anti-sense strands listed herein, respectively. In various aspects, the first and second strands are the anti-sense and sense strands listed herein, respectively.

[0060] In one aspect, the sense strand is about 30 or fewer nucleotides (nt) in length.

[0061] In one aspect, the antisense strand is about 30 or fewer nucleotides in length.

[0062] In one aspect, the antisense strand forms a duplex region with a sense strand, wherein the duplex region is about 15 to 30 nucleotide pairs in length. [0063] In one aspect, the antisense strand is about 15 to about 30 nucleotides in length, including 18, about 18, and about 19 to about 23 nucleotides in length. In one aspect, the antisense strand has at least the length selected from about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, 18 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 2 1 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides and 30 nucleotides. [0064] In one aspect, the RNAi agent comprises a modification that causes the RNAi agent to have increased stability in a biological sample or environment (e.g., cytoplasm, interstitial fluid, blood serum, or lung or intestinal lavage).

[0065] In one aspect, the RNAi agent comprises at least one sugar backbone modification (e.g., phosphorothioate linkage) or at least one 2'-modified nucleotide.

[0066] In one aspect, the RNAi agent comprises: at least one 5'-uridine-adenine-3' (S ua s') dinucleotide, wherein the uridine is a 2'-modified nucleotide; at least one 5'-uridine-guanine- 3' (5'-ug-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; at least one 5'- cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide. These dinucleotide motifs are particularly prone to serum nuclease degradation (e.g. RNase A). Chemical modification at the 2'-position of the first pyrimidine nucleotide in the motif prevents or slows down such cleavage. This modification recipe is also known under the term 'endo light'.

[0067] In one aspect, the RNAi agent comprises a 2'-modification selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl (2'-OMe or 2'OMe), 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0- dimethylaminopropyl (2 -O-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), and 2'-0-

N-methylacetamido (2'-0-NMA). In one aspect, all pyrimidines (uridine and cytidine) are 2' O- methyl-modified nucleosides. [0068] In one aspect, the RNAi agent comprises at least one blunt end.

[0069] In one aspect, the RNAi agent comprises an overhang having 1 nt to 4 nt unpaired.

[0070] In one aspect, the RNAi agent comprises an overhang at the 3'-end of the antisense strand of the RNAi agent. [0071] In one aspect, the RNAi agent is ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin. [0072] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 50% in Huh-7 cells in nude mice.

[0073] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 70% at a concentration of 10 nM in Huh-7 cells in vitro.

[0074] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 75% at a concentration of 10 nM in Huh-7 cells in vitro.

[0075] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 80% at a concentration of 10 nM in Huh-7 cells in vitro.

[0076] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 90% at a concentration of 10 nM in Huh-7 cells in vitro.

[0077] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 95% at a concentration of 10 nM in Huh-7 cells in vitro.

[0078] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 99% at a concentration of 10 nM in Huh-7 cells in vitro.

[0079] In one aspect, the RNAi has an EC50 of no more than about 0.1 nM nM in Huh-7 cells in vitro. EC50 is effective concentration to reduce gene expression by 50%. [0080] In one aspect, the RNAi has an EC50 of no more than about 0.01 nM nM in Huh-7 cells in vitro. [0081] In one aspect, the RNAi has an EC50 of no more than about 0.001 nM nM in Huh-7 cells in vitro.

[0082] An RNAi agent comprising a sense and antisense strand of an RNAi described herein.

[0083] In one particular specific aspect, the present disclosure relates to a composition comprising a Hepcidin RNAi agent comprising a first strand and a second strand, wherein the sequence of the first strand and/or second strand comprise at least 15 contiguous nucleotides, differing by 0, 1, 2 , or 3 nucleotides from the sequence of the first and/or second strand of a RNAi agent to Hepcidin selected from the specific duplexes provided herein. [0084] In one particular specific aspect, the present disclosure relates to a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the sequence of the first strand and/or second strand comprise the sequence of the first and/or second strand, respectively, of a RNAi agent to Hepcidin selected from the specific duplexes provided herein. [0085] In one particular specific aspect, the present disclosure relates to a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the sequence of the first strand and/or second strand are the sequence of the first and/or second strand, respectively, of a RNAi agent to Hepcidin selected from the specific duplexes provided herein. [0086] In one particular specific aspect, the present disclosure relates to a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the sequence of the first strand and/or second strand are the sequence of the first and/or second strand, respectively, of a RNAi agent to Hepcidin selected from the specific duplexes provided herein, wherein the sequence of the first and/or second strand further comprise a terminal dinucleotide. [0087] In one particular specific aspect, the present disclosure relates to a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the sequence of the first strand and/or second strand are the sequence of the first and/or second strand, respectively, of a RNAi agent to Hepcidin selected from the specific duplexes provided herein, wherein the sequence of the first and/or second strand further comprise a terminal UU dinucleotide. [0088]

[0089] In one particular specific aspect, the present disclosure relates to a composition comprising a Hepcidin RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and antisense strand comprise at least 15 contiguous nucleotides differing by

0, 1, 2, or 3 nucleotides, from the sense and antisense strand, respectively, of an RNAi agent to Hepcidin selected from the specific duplexes provided herein. [0090] Various particular specific aspects of this aspect are described below.

[0091] In one aspect, the composition comprises a second RNAi agent to Hepcidin. In various aspects, the second RNAi agent is physically separate from the first, or the two are physically connected (e.g., chemically linked or otherwise conjugated). In some aspects, the first and second RNAi agents are combined within the same composition (e.g., both in the same lipid nanoparticle).

[0092] In one aspect, the antisense strand is about 30 or fewer nucleotides in length.

[0093] In one aspect, the sense strand and the antisense strand form a duplex region about 15 to about 30 nucleotide pairs in length. [0094] In one aspect, the antisense strand is about 15 to about 36 nt in length including about 18 to about 23 nt in length, and including about 19 to about 2 1 nt in length and about 19 to about 23 nt in length. In one aspect, the antisense strand has at least the length selected from about 15 nt, about 16 nt, about 17 nt, 18, about 18 nt, about 19 nt, about 20 nt, about 2 1 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt and about 30 nt.

[0095] In one aspect, the RNAi agent comprises a modification that causes the RNAi agent to have increased stability in a biological sample or environment.

[0096] In one aspect, the RNAi agent comprises a modified sugar backbone such as, e.g., a phosphorothioate linkage, or comprises a 2'-modified nucleotide.

[0097] In one aspect, the RNAi agent comprises: at least one 5'-uridine-adenine-3' (5'-ua- 3') dinucleotide, wherein the uridine is a 2'-modified nucleotide; at least one 5'-uridine-guanine- 3' (5'-ug-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; at least one 5'- cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; or at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide.

[0098] In one aspect, the RNAi agent comprises a 2'-modification selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2 -O-MOE), 2'-0- aminopropyl (2 -O-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2 - O-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), and 2'-0-N-methylacetamido (2 -

O-NMA). In one aspect, all pyrimidines (uridine and cytidine) are 2' O-methyl-modified nucleosides. [0099] In one aspect, the RNAi agent comprises at least one blunt end.

[00100] In one aspect, the RNAi agent comprises an overhang having 1 to 4 nt unpaired.

[00101] In one aspect, the RNAi agent comprises an overhang at the 3'-end of the antisense strand of the RNAi agent.

[00102] In one aspect, the RNAi agent is ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin. [00103] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 50%, 60%, 70% or 80% in Huh-7 cells in nude mice. [00104] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 70% at a concentration of 10 nM in Huh-7 cells in vitro.

[00105] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 80% at a concentration of 10 nM in Huh-7 cells in vitro.

[00106] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 90% at a concentration of 10 nM in Huh-7 cells in vitro.

[00107] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 95% at a concentration of 10 nM in Huh-7 cells in vitro.

[00108] In one aspect, the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 99% at a concentration of 10 nM in Huh-7 cells in vitro.

[00109] In one aspect, the RNAi has an EC50 of no more than about 0.1 nM in Huh-7 cells in vitro.

[00110] In one aspect, the RNAi has an EC50 of no more than about 0.01 nM in Huh-7 cells in vitro.

[00111] In one aspect, the RNAi has an EC50 of no more than about 0.001 nM in Huh-7 cells in vitro.

[00112] A method of treatment using a composition comprising a RNAi agent described herein.

[00113] In one particular specific aspect, the present disclosure relates to a method of treating a Hepcidin-related disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition comprising an RNAi agent comprising an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nucleotides from the antisense strand of an RNAi agent to Hepcidin selected from those specific duplexes provided above and as listed in any of Tables 1 to 4 and 6 . In one aspect, the RNAi agent to Hepcidin comprises an antisense strand duplexed with a sense strand, wherein the sense and antisense strands are selected from one or more of the sequences provided in any of Tables 1 to 4 and 6 . [00114] Various particular specific aspects of this aspect are described below. Any aspects disclosed herein that are not mutually exclusive can be combined.

[00115] In one aspect, the present disclosure relates to such a method, wherein the composition comprising a RNAi agent further comprises a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nucleotides from the sense strand of a RNAi agent to Hepcidin selected from the specific duplexes provided herein and as listed, e.g., in any Table herein.

[00116] In one aspect of the method, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti-sense strand is the sequence of the sense and/or the anti-sense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in Table 1, wherein the composition further comprises a pharmaceutically effective formulation.

[00117] In one aspect of the method, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti-sense strand comprises the sequence of the sense and/or the anti-sense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in any of Tables 1 to 4 and 6, wherein the composition further comprises a pharmaceutically effective formulation.

[00118] In one aspect, the disclosure pertains to a method of inhibiting expression of Hepcidin in a subject, wherein the method comprises the step of administering to the subject an effective amount of a composition comprising an RNAi agent to Hepcidin (e.g., any described herein). In one aspect, the subject is human. In one aspect, the subject has a Hepcidin-related disease. In one aspect, the Hepcidin-related disease is anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and/or hair loss. In some embodiments, the subject is a dialysis patient. In some embodiments, the Hepcidin-related disease is anemia and the subject is a dialysis patient. [00119] In one aspect, the method further comprises the step of administering an additional treatment.

[00120] In one aspect, the additional treatment is a method (or procedure). In one aspect, the additional treatment is a therapeutically effective dose of a composition. [00121] In one aspect, the additional treatment and the RNAi agent can be administered in any order, or can be administered simultaneously.

[00122] In one aspect, the method further comprises the step of administering an additional treatment for a Hepcidin-related disease.

[00123] In one aspect, the method further comprises the step of administering an additional treatment. A RNAi agent to Hepcidin can be used in conjunction with any additional treatment disclosed herein, as appropriate for the disease, optionally, in further conjunction with one or more additional RNAi agents to Hepcidin. [00124] It will be understood that references to any additional treatment are meant to also include the pharmaceutically acceptable salts of any of the active substances. If active substances comprised by components (a) and/or (b) have, for example, at least one basic center, they can form acid addition salts. Corresponding acid addition salts can also be formed having, if desired, an additionally present basic center. Active substances having an acid group, e.g., COOH, can form salts with bases. The active substances comprised in components (a) and/or (b) or a pharmaceutically acceptable salts thereof may also be used in form of a hydrate or include other solvents used for crystallization.

[00125] In one aspect, the composition comprises a second RNAi agent to Hepcidin. In various aspects, the second RNAi agent is physically distinct from the first, or the two are physically connected (e.g., linked or conjugated). In some aspects, the first and second RNAi agents are combined within the same composition (e.g., both in the same lipid nanoparticle).

[00126] A method of inhibiting the expression of Hepcidin, using an RNAi comprising an RNAi agent described herein. [00127] In one particular specific aspect, the present disclosure relates to a method of inhibiting the expression of Hepcidin in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition comprising an RNAi agent of the disclosure. In one aspect, the RNAi comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of an RNAi agent to Hepcidin selected from those specific duplexes provided above and as listed in any of Tables 1 to 4 and 6.

[00128] In one aspect of the method, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti-sense strand is the sequence of the sense and/or the anti-sense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in any of Tables 1 to 4 and 6, wherein the composition is in a pharmaceutically effective formulation.

[00129] In one aspect of the method, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti-sense strand comprises the sequence of the sense and/or the anti-sense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in any of Tables 1 to 4 and 6, wherein the composition is in a pharmaceutically effective formulation. [00130] Various particular specific aspects of this aspect are described below.

[00131] In one aspect, the individual is afflicted with or susceptible to an Hepcidin-related disease.

[00132] In one aspect, the Hepcidin-related disease is anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and/or hair loss. In some embodiments, the subject is a dialysis patient. In some embodiments, the Hepcidin-related disease is anemia and the subject is a dialysis patient.

[00133] In one aspect, the method further comprises the step of administering an additional treatment.

[00134] In one aspect, the additional treatment and the RNAi agent can be administered in any order or can be administered simultaneously.

[00135] In one aspect, the method further comprises the step of administering an additional treatment for anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and/or hair loss. In some embodiments, the subject is a dialysis patient. In some embodiments, the Hepcidin-related disease is anemia and the subject is a dialysis patient. [00136] In one aspect, the composition comprises a second RNAi agent to Hepcidin. In various aspects, the second RNAi agent is physically separate from the first, or the two are physically connected (e.g., covalently linked or otherwise conjugated). In some aspects, the first and second RNAi agents are combined within the same composition (e.g., both in the same lipid nanoparticle).

[00137] In one aspect, the method further comprises the step of administering an additional RNAi agent which comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nucleotides from the antisense strand of a RNAi agent to Hepcidin selected from the specific duplexes provided herein and as listed, e.g., in any Table herein.

[00138] Pharmaceutical compositions of a RNAi agent to Hepcidin [00139] In one particular specific aspect, the present disclosure relates to a composition comprising a RNAi agent of the present disclosure. In one aspect, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the anti-sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nucleotides from the anti-sense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in any of Tables 1 to 4 and 6, wherein the composition is in a pharmaceutically effective formulation.

[00140] In one aspect, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti- sense strand is the sequence of the sense and/or the anti-sense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in any of

Tables 1 to 4 and 6, wherein the composition is in a pharmaceutically effective formulation. [00141] In one aspect, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the sequence of the sense and/or anti- sense strand comprises the sequence of the sense and/or the anti-sense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in any of Tables 1 to 4 and 6, wherein the composition is in a pharmaceutically effective formulation. [00142] In one aspect, the present disclosure pertains to the use of a RNAi agent in the manufacture of a medicament for treatment of a Hepcidin-related disease, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent to Hepcidin selected from those specific duplex provided herein and as listed, e.g., in any Table herein.

[00143] Specific Aspects of RNAi Agents to Hepcidin comprising mismatches from the disclosed sequences [00144] Various specific aspects of a RNAi agent to Hepcidin are disclosed herein. The present disclosure encompasses the example modified variants provided in any of Tables 1 to 4 and 6, and the corresponding unmodified sequences and other modified variants. Specific aspects of the present disclosure include RNAi agents which comprise sequences differing by 0,

1, 2 , or 3 nt (nucleotides) or bp [basepair(s)] (e.g., with 0, 1, 2 or 3 mismatches) from any of the RNAi agents listed in any of Tables 1 to 4 and 6, and modified and unmodified variants thereof. As described in additional detail below, a mismatch is defined herein as a difference between the base sequence (e.g., A instead of G) or length when two sequences are maximally aligned and compared. In addition, as described in more detail below, an "unmodified variant" is a variant in which the base sequence is identical, but none of the bases are modified; this includes, for example, a sequence identical to the corresponding portion of the wild-type Hepcidin mRNA or gene. A "modified variant" contains one or more modifications (or one or more fewer or different modifications) to a nucleotide, sugar, phosphate or backbone, and/or addition of one or more moieties; but without a change, substitution, addition, or deletion to the base sequence. A particular sequence and its modified or unmodified variants have 0 mismatches among them. [00145] In one particular aspect, the present disclosure comprises a RNAi agent comprising a sense and an anti-sense strand, wherein the sense and/or anti-sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of the sense and/or anti-sense strand of: any of the RNAi agents listed in any of Tables 1 to 4 and 6, and modified and unmodified variants thereof. [00146] In another particular aspect, the RNAi agent comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand of any of the RNAi agents listed in any of Tables 1 to 4 and 6, and modified and unmodified variants thereof. [00147] Other aspects [00148] Various particular specific aspects of this disclosure are described below. Any aspects disclosed herein that are not mutually exclusive can be combined.

[00149] In one aspect, the disclosure pertains to a composition according to any of the above aspects, for use in a method of treating a Hepcidin-related disease in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a composition according to any of the claims. [00150] Various particular specific aspects of this aspect are described below.

[00151] In one aspect, the disclosure pertains to the composition according to any of the above aspects, for use in a method of inhibiting the expression of Hepcidin in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a composition according to any of the above aspects. [00152] One aspect of the disclosure is the use of a composition according to any of the above aspects, in the manufacture of a medicament for treatment of an Hepcidin-related disease.

[00153] In one aspect, the Hepcidin-related disease is selected from any disease listed herein.

[00154] In one aspect, the disclosure pertains to the composition of any of the above aspects, for use in the treatment of an Hepcidin-related disease.

[00155] In one aspect, the Hepcidin-related disease is selected from any disease listed herein.

[00156] In one aspect, the disclosure relates to a method of inhibiting the expression of Hepcidin in an cell, comprising the step of introducing into the cell a composition comprising an RNAi agent comprising an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of an RNAi agent to Hepcidin selected from the Hepcidin siRNAs disclosed herein.

[00157] In one aspect, the disclosure relates to a method of inhibiting the expression of Hepcidin in an cell, comprising the step of introducing into the cell a composition comprising an RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand, and the sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sense strand of an RNAi agent to Hepcidin selected from the Hepcidin siRNAs disclosed herein.

[00158] Definitions

[00159] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[00160] Hepcidin

[00161] By "Hepcidin" is meant the gene or its protein product, a peptide hormone, also known as: HAMP (Hepcidin anti-microbial protein or peptide); HEPC; HFE2B; LEAP1 (LEAP-1); PLTR; OMIM: 606464; HomoloGene: 81623; GeneCards: HAMP Gene; Entrez 57817; EnsembI ENSG000001 05697; UniProt P81 172 ; RefSeq (mRNA) NM_021 175; RefSeq (protein) NP_066998; Location (UCSC) Chr 19: 35.77 - 35.78 Mb. Krause et al. FEBS Lett. 480: 147- 150; and Pigeon et al. 2001 J. Biol. Chem. 276: 781 1-9. See also: Ganz 2003 Blood 102: 783- 8; Roy et al. 2005 Curr. Opin. Hemat. 12: 107-1 11; Fleming et al. 2006 Semin. Liver Dix. 25:

4 1 1-9; Park et al. 2001 J. Biol. Chem. 276: 7806-10; Majore et al. 2002 Haematologica 87: 221- 2; Kluver et al. 2002 J. Pept. Res. 59 : 241-8 ; Hunter et al. 2002 J. Biol. Chem. 277 : 37597-

603 ; Weinstein et al. 2003 Blood 100 : 3776-81 ; Nemeth et al. 2003 Blood 101 : 2461-3; Roetto et al. 2003 Nat. Genet. 33: 21-2; Strausberg et al. 2003 Proc. Natl. Acad. Sci USA 99: 16899- 903; Gehrke et al. 2003 Blood 102: 371-6; Merryweather-Clarke et al. 2004 Human Mol. Genet. 12:2241-7; Clark et al. 2003 Genome Res. 13: 2265-70; Roetto et al. 2004 Blood 103: 2407-9; Jacolot et al. 2004 Blood 103: 2835-40; and Ota et al. 2004 Nat. Genet. 36: 40-45. [00162] A recent gene duplication event in mouse evolution has led to the presence of two similar hepcidin genes in mice, Hepcidinl and Hepcidin2. Ilyin et al. 2003 FEBS Lett. 542: 22- 26. Mouse hepcidin2 lacks several conserved residues found in mammalian hepcidins. Lou et al. 2004 Blood 103: 2816-2821 .

[00163] The Hepcidin gene product is involved in the maintenance of iron homeostasis, and it is necessary for the regulation of iron storage in macrophages, and for intestinal iron absorption. These peptides exhibit antimicrobial activity. [00164] The preproprotein (or preprohormone or preprohepcidin) (84 aa) and proprotein (or prohormone or prohepcidin) (60 aa) are processed into mature peptides of 20, 22 and 25 amino acids. The 25-aa peptide is secreted mainly by the liver and is considered the "master regulator" of iron metabolism. The 20- and 22-aa metabolites exist in the urine. The N-terminal region of Hepcidin is required for function; deletion of the 5 N-terminal amino acids results in loss of function.

[00165] The active Hepcidin peptides are rich in cysteines, which form intramolecular bonds that stabilize their beta sheet structures. [00166] Hepcidin is mainly synthesized in the liver, with smaller amounts found to be synthesized in other tissues. Bekri et al. 2006 Gastroent. 131 : 788-96. [00167] Hepcidin Functions: Iron metabolism. The 25-aa peptide is secreted mainly by the liver and is considered the "master regulator" of iron metabolism. [00168] Hepcidin inhibits iron transport by binding to the iron export channel ferroportin, which is located on the basolateral surface of gut enterocytes and the plama membrane of reticuloendothelial cells (macrophages). By inhibiting ferroportin, hepcidin prevents enterocytes of the intestines from secreting iron ito the hepatic portal system, thereby functionally reducing iron absorption. The iron release from macrophages is also prevented by ferroportin inhibition; therefore, the hepcidin maintains iron homeostasis. Hepcidin activity is also partially responsible for iron sequestration seen in anemia of chronic inflammation such as inflammatory bowel disease, chronic heart failure, carcinomas, rheumatoid arthritis and renal failure. [00169] Mutations in the hepcidin gene cause hemochromatosis type 2B, also known as juvenile hemochromatosis, a disease caused by severe iron overload that results in cardiomyopathy, cirrhosis, and endocrine failure. The majority of juvenile hemochromatosis cases are due to mutations in hemojuvelin, a regulator of hepcidin production. [00170] Genetically modified mice engineered to overexpress hepcidin die shortly after birth with severe iron deficiency, suggesting a central and not redundant role in iron regulation. The first evidence that linked hepcidin to anemia of inflammation came when researchers examined tissues from two patients with liver tumors with a severe microcytic anemia that did not respond to iron supplements. The tumor tissue overproduced hepcidin, and removing the tumors surgically cured the anemia. [00171] There are many diseases wherein failure to adequately absorb iron contributes to iron deficiency and iron deficiency anemia. The treatment will depend on the hepcidin levels, as oral treatment will likely be ineffective if hepcidin is blocking enteral absorption. [00172] Hepcidin-related diseases [00173] siRNAs to Hepcidin can be used to treat Hepcidin-related diseases. A "Hepcidin- related disease" is any disease associated with Hepcidin and/or a mutation and/or an over- expression of a wild-type and/or mutant Hepcidin, and/or diseases wherein disease progression is enhanced by or prognosis worsened by the presence of Hepcidin and/or a mutation and/or an over-expression of wild-type and/or mutant Hepcidin. Non-limiting examples of Hepcidin-related diseases include: anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and/or hair loss. In some embodiments, the subject is a dialysis patient. In some embodiments, the Hepcidin-related disease is anemia and the subject is a dialysis patient. The prevalence of iron and ESA-refractory anemia is high in chronic hemodialysis population.

[00174] In various aspects, the present disclosure pertains to the use of Hepcidin RNAi agents in inhibiting and/or reducing the level and/or activity of Hepcidin, for treatment of Hepcidin-related diseases, particularly those diseases in which the level and/or activity of Hepcidin is excessive. Such diseases include anemia and related diseases, including EPO- resistant anemia (e.g., EPO-resistant anemia in end-stage renal disease or ESRD). These include: anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia. Ganz et al. 2003 Blood 102: 783-788; Yuan et al. 2003 Ann. Med. 35: 578-591 ; O'Neil et al. 2005 Sem.

Liver Dis. 25: 381-391; and Moos et al. 2004 Ann. New York Acad. Sci. 1012: 14-26. In some cases, anemia can be defined as a decrease of hemoglobin (Hb) of 1 g/dl or more. Sasu et al. 2010 Blood 115: 3616-24. Inflammatory hepcidin induction is accompanied by impaired dietary iron uptake, iron sequestration and anemia.

[00175] Anemia is common in patients who have both heart failure and chronic kidney disease (CKD or cardiorenal syndrome), and there is an association between anemia and progression of both diseases. In cardiorenal-anemia syndrome, anemia and cardiac and renal dysfunction form a vicious circle, with each constituent capable of causing or worsening the other two. van der Putten 2008 Nat. Clin. Pract. 4 : 47-57. [00176] Causes of anemia in patients with CKD also include infection and absolute iron deficiency. Eckhardt 2000 Clin. Nephrol. 53 (Suppl. 1): S2-S8. Blood loss is a common cause of anemia in patients with renal failure. This blood loss include occult gastrointestinal blood loss, blood being retained in extracorporeal circuits during dialysis, and withdrawal of blood for laboratory tests. Hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies can also cause anemia in patients with CKD. Eckhardt 2000; Drueke et al. 2002 Nephrol. Dial. Transplant 17 (Suppl. 5): S28-S31 ; and de Silva et al. 2006 Am. J. Cardiol. 98: 391-398. Anemia can also sometimes be associated with cancer, chemotherapy, AIDS (including enamia due to tretment with zidovudine), and surgical procedures van der Putten 2008 Nat. Clin. Pract. 4 : 47-57.

[00177] Additional Hepcidin-related diseases include runted growth, hair loss and iron- deficient erythropoiesis. In some cases, Hepcidin mediates resistance to supraphysiological levels of erythropoiesis stimulating agent (ESA) (Epoetin alfa or darbepoetin alfa). Zimmerman et al. 1999 Kidney Internat. 55: 648-658; Yeun et al. Am. J. Kidney Dis. 35: 469-476; Horl et al. 2000 Neph. Dial. Transplant. 15 Suppl. 4 : 43-50; Barany et al. 1997 Am. J. Kidney Diseases 29: 565-568; Gudbjornsson et al. 1992 Ann. Rheumatic Dis. 5 1: 747-752; and Nordstrom et al. 1997 Rheumat. Internat. 17: 67-73. Over-expression of hepcidin in mice for four months caused hypoferremia, decreased size and substantial hair loss. Sasu et al. 2010 Blood 115: 3616-24. [00178] Thus, the present disclosure pertains to the use of Hepcidin RNAi agents to treat a Hepcidin-related disease (e.g., as listed herein) in a patient. [00179] Thus, the present disclosure encompasses Hepcidin RNAi agents and the uses thereof for Hepcidin-related diseases.

[00180] USE OF HEPCIDIN RNAi AGENTS TO TREAT HEPCIDIN-RELATED DISEASES

[00181] In various aspects, the present disclosure pertains to the use of Hepcidin RNAi agents, which inhibit and/or decrease the level and/or activity of Hepcidin, to treat Hepcidin- related diseases. [00182] In earlier studies, inhibiting or decreasing the level and/or activity of Hepcidin have been shown to be effective in treatment of Hepcidin related diseases. [00183] In one study, two anti-hepcidin shRNAs decreased mHep liver mRNA and serum hepcidin in mice treated with Brucella abortus (an anemia model). Hepcidin suppression resulted in a significant increase in mean cell volume (MCV) and mean cell hemoglobin (MCH). Both moderate and profound hepcidin suppression resulted in an increase in serum iron compared to controls; these data suggest that hepcidin suppression mobilized iron effectively in animals with inflammation. Sasu et al. 2010 Blood 115: 3616-24.

Hepcidin RNAi agent for use in treating various Hepcidin-related diseases [00184] In one aspect, the Hepcidin RNAi agent of the present disclosure comprises a sequence disclosed herein and is administered to a patient in need thereof (e.g., a patient suffering from an Hepcidin-related disease disclosed herein or known in the literature). In one aspect, the Hepcidin RNAi agent of the present disclosure is administered to a patient in need thereof, along with one or more additional pharmaceutical agent appropriate for that disease. For example, a patient suffering from an Hepcidin-related disease can be administered a pharmacologically effective amount of one or more Hepcidin RNAi agent along with a pharmacologically effective amount of one or more of any Hepcidin-related disease treatment listed herein, and/or any other Hepcidin-related disease treatment known in the art. [00185] A patient suffering from a Hepcidin-related disease can be administered one or more RNAi agent to Hepcidin and one or more additional Hepcidin-related disease treatment. This additional treatment can be selected from the list of any disease treatment listed herein, and/or any anti- Hepcidin-related disease treatment known in the art. [00186] The Hepcidin RNAi agents of the instant disclosure can be administered along with (as part of the same therapeutic treatment regimen, prior to, simultaneously with, or after) one or more additional therapeutics to treat a Hepcidin-related disease. These one or more additional therapeutics can be selected from: iron administration, chelation therapy, phlebotomy, erythropoiesis stimulating agent (ESA) (e.g., Epoetin alfa or darbepoetin alfa), anti-Hepcidin antibody, hemodialysis, and hyperbaric oxygen. [00187] In combination with ESA, moderate hepicidin suppression mediated by a shRNA resulted in significant increase in Hb and a complete correction of anemia. Sasu et al. 2010 Blood 115: 3616-24. [00188] The patient can also be administered more than one RNAi agent to Hepcidin. [00189] In the case of Hepcidin-related diseases, the RNAi agent(s) and additional disease treatment(s) can be administered in any order, simultaneously or sequentially, or in multiple doses over time. Administration of the RNAi agent and the additional treatment can be, for example, simultaneous, concurrent, separate or sequential. [00190] Simultaneous administration may, e.g., take place in the form of one fixed combination with two or more active ingredients, or by simultaneously administering two or more active ingredients that are formulated independently. Sequential use (administration) preferably means administration of one (or more) components of a combination at one time point, other components at a different time point, that is, in a chronically staggered manner, preferably such that the combination shows more efficiency than the single compounds administered independently (especially showing synergism). Separate use (administration) preferably means administration of the components of the combination independently of each other at different time points, preferably meaning that the components (a) and (b) are administered such that no overlap of measurable blood levels of both compounds are present in an overlapping manner (at the same time). [00191] Also combinations of two or more of sequential, separate and simultaneous administration are possible, preferably such that the combination component-drugs show a joint therapeutic effect that exceeds the effect found when the combination component-drugs are used independently at time intervals so large that no mutual effect on their therapeutic efficiency can be found, a synergistic effect being especially preferred. [00192] The term "delay of progression" as used herein means administration of the combination to patients being in a pre-stage or in an early phase, of the first manifestation or a relapse of the disease to be treated, in which patients, e.g., a pre-form of the corresponding disease is diagnosed or which patients are in a condition, e.g., during a medical treatment or a condition resulting from an accident, under which it is likely that a corresponding disease will develop. [00193] "Jointly therapeutically active" or "joint therapeutic effect" means that the compounds may be given separately (in a chronically staggered manner, especially a sequence-specific manner) in such time intervals that they preferably, in the warm-blooded animal, especially human, to be treated, still show a (preferably synergistic) interaction (joint therapeutic effect). Whether this is the case, can inter alia be determined by following the blood levels, showing that both compounds are present in the blood of the human to be treated at least during certain time intervals.

[00194] Methods of identifying Hepcidin RNAi agents [00195] Any method known in the art can be used to identify efficacious Hepcidin RNAi agents useful for treating Hepcidin-related diseases. A few non-limiting examples of such methods are disclosed herein. Any one or more of the following, in addition to other methods known in the art, can be used. [00196] For example: In various steps, Hepcidin RNAi agents are tested for efficacy and tolerance to modifications and modification from 19-mer to 18-mer format. [00197] For example: Hepcidin RNAi candidates can be tested by target knockdown in Huh7 cells and primary hepatocytes. This can be tested using any method known in the art, including but not limited to RNAi Max. Preferably, the RNAi candidate demonstrates at least about 80% mRNA knockdown at 48 hours. The mechanism can optionally be confirmed to be RNAi (RNA interference) by identifying specific cleavage products using RACE. [00198] Cross-reactivity with cyno monkeys can be assessed in primary cyno hepatocytes at 48 hours. Optionally, the IC50 can be within five-fold of human siRNA activity. [00199] Off-target effects of the candidate Hepcidin RNAi agent can be assessed. For example, cell growth in non-hepcidin-expressing cells can be tested. Preferably, there is no effect relative to, for example, YFP siRNA. [00200] Immune stimulation by the candidate RNAi agent can be tested using whole blood assay. Preferably, there would be no detectable activation of cytokine expression. [00201] Toxicity can also be assessed. MTD (maximum tolerated dosage) can be assessed in mice. A Mini-tox study can be performed for 2 weeks. Optionally, a conjugate can be used. [00202] Efficacy in animal models can be assessed. For example, a conjugate can be used to deliver mouse hepcidin siRNA to liver and effectively knockdown the target. Preferably, the mRNA knockdown will be at least about 80% at 96 hours. [00203] Efficacy in cyno monkeys can also be tested. [00204] PK (pharmacokinetics) and bio-distribution of formulated RNAi agents can be tested, for example, in rodents, using, for example, LNP (lipid nanoparticle)-formulated RNAi agents. Preferably, the RNAi targets the liver.

[00205] In addition, optionally PK analysis can be done of naked RNAi agents in anephric/kidney-impaired rodent model. [00206] Efficacy in the Brucella abortus model can also be tested. This mouse model for Al involves administration of heat-inactivated Brucella abortus (BA). C57BL/6 mice treated with a single dose of BA developed anemia within one week. Sasu et al. 2010 Blood 115: 3616-24.

[00207] In vivo experiments can also be performed using knock-in mice, wherein the mouse mHep locus or both the mouse mHepd and mHepc2 loci are replaced by the human hHepc locus. Sasu et al. 2010 Blood 115: 3616-24. [00208] These examples are meant to be non-limiting examples of methods that can be used to test candidate or potential Hepcidin RNAi agents. [00209] These and other methods known in the art and/or known or available to one of ordinary skill can be used to test candidate Hepcidin RNAi agents.

[00210] Hepicidin RNAi agent candidate chemistry optimization [00211] In particular, several steps can be performed for optimizing the chemistry of the lead(s) Hepcidin RNAi agent(s): [00212] Hepcidin sequences are identified, including 6 lead sequences (397, 398, 400, 401, 402 and 403). Serum stability highlights the 5' terminal U as a fragile site. [00213] Tolerance for chemical modification is assessed. Hepcidin sequences 400 and 402 appear to be the most tolerant for chemical modification. Indirect evidence shows that 5' dT vs 5'U does stabilize the 5' end of the guide strand. No "more modified" format did improve potency. [00214] The 3' overhang is optimized, e.g., PAZ ligands are screened. Test sequences are hs_HAMP_ 400 and 402. The parent stem format is A106S42. 17 PAZ ligands are tested on the guide strand only +/- ribitol, +/- MOE clamp (wherein the first 2 nt on the 3' end have 2'- MOE). [00215] Passenger strand optimization is done. For example, this was done using the assymetrical design. [00216] Internal modifications for stabilization are assessed. For example, chemical stabilization at the cleavage sites is tested. [00217] These and other tests available or known to one of ordinary skill in the art can be used to evaluate and modify the chemistry of a candidate Hepcidin RNAi agent.

[00218] Hepcidin RNAi Agent candidate selection [00219] The instant disclosure pertains to RNAi agents to Hepcidin for use in treating Hepcidin-related diseases. These agents have various structural properties, including having an 18-mer sequence. Optionally, the agent is modified, including but not limited to, modifications of one or more sugar or phosphate, and with, in 3' order, a linker and a 3' end cap. The 5' and/or 3' end of the sense strand can be conjugated to other components which increase targeting to and/or uptake by the liver. [00220] The disclosure also pertains to compositions comprising the RNAi agent to Hepicidin and, for example, an excipient or pharmaceutically acceptable carrier. [00221] The Hepcidin mRNA is unusually short, thus leaving only a small room for siRNA design, and very small room for pan-specific siRNAs (those which target both humans and one or more test animals). [00222] In this work, using the human sequence, 419 possible 19-mer Hepcidin RNAi agents were identified. [00223] A subset of these were selected and tested. These sequences are shown below in Table 1 (as DNA 19-mers) and Table 2 (as RNA 19-mers).

[00224] TABLE 1. 19-MER TARGET SEQUENCES (DNA)

[00225] One of ordinary skill in the art can prepare 18-mer sequences from the 19-mer sequences disclosed above, e.g., by using nt 1-18 or 2-19 of the various sequences. For example, nt 1-18 of a guide strand, above, can be paired with nt 2-19 of the corresponding sense strand to form a blunt-ended duplex. In addition, nt 2-1 9 of the guide strand can be paired with nt 1-18 of the corresponding sense strand to form a blunt-ended duplex. [00226] 6 potent sequences were identified: 397, 398, 400, 401 , 402, and 403. These top candidates are shown below, as 18-mers, in Table 3 .

TABLE 3. TOP 6 CANDIDATES. 18-mer SEQUENCES

[00227] The sequences of these 6 leads candidates is also shown below, compared to that in various species, including human, Macaca, Rhesus, cynomolgus, mouse (two sequences), and rat. In the sequences shown above, from top to bottom: human_RE (REF or reference) is SEQ ID NO: 155; Macaca_f (cyno) is SEQ ID NO: 156; Rhesus m (monkey) is SEQ ID NO: 157; an alternative cyno (cynomolgus monkey) sequence is SEQ ID NO: 158; two mouse sequences are SEQ ID NO: 159 and 160; rat is SEQ ID NO: 161 ; and human hepcidin RNAi agents are: 397 (SEQ ID NO: 162); 398 (SEQ ID NO: 163); 400 (SEQ ID NO: 164); 401 (SEQ ID NO: 165); 402 (SEQ ID NO: 166), and 403 (SEQ ID NO: 167). The sequences shown are sense strand. [00228] The efficacy of the lead sequences is shown in Fig. 1 and 6. Various Hepcidin RNAi agent sequences were tested, but many were not efficacious, or so poorly efficacious as to not be suitable for therapeutic use. [00229] This disclosure notes that there is a single mismatch between the human and cyno sequences, but this is not in the seed region. Various experiments showed that this one mismatch did not greatly alter RNAi agent activity in human and cyno cells; the cyno sequence performed well in human cells, for example.

[00230] Screening of Human (hs or Has) Hepcidin RNAi agents

[00231] 2 1 human Hepcidin RNAi agents are screened as both 19-mers and 18-mers. The human Hepcidin iRNAs are also sometimes designated "hs" or "Hsa" for Homo sapiens. Some data are shown in Fig. 6. [00232] Of these human Hepcidin RNAi agents, the top candidates were selected: 398, 400, 401, 402 and 403, each representing an efficacious RNAi agent. [00233] Variants of these various human Hepcidin RNAi agents were created. The efficacy of some are shown in Fig. 2A and 2B. These variously have a MOE clamp, a ribitol spacer (or no spacer), and a 3' end cap (C6) or a 3' dinucleotide. 18-mer and 19-mer formats are included. [00234] 9 different stem chemistries were tested on the top 6 human siRNAs. Results are shown in Fig. Fig. 2A and 2B. The results show that 400, 402 and 403 18-mers tolerate heavy stem chemistry modification. In these experiments, among the molecules tested, the most active versions of 400 and 402 comprised a 2'-MOE clamp (wherein the last two nucleotides on the 3' end of each strand are 2'-MOE), and wherein 3' end of the antisense strand terminates in a phosphate (designated herein as "p" or "PO") and further comprises a spacer (ribitol), a phosphate and a 3' end cap. [00235] HEPCIDIN 400 and 402 RNAi AGENTS [00236] Of the 6 lead candidates, two sequences, hs HAMP (human Hepcidin) 400 and 402 (of SEQ ID NOs: 87 and 92; and SEQ ID NOs: 89 and 94, respectively), showed particularly high tolerance to modifications of chemistry. Many successful modified variants were created with these sequences. Several formats, including those of 18-mer, 19-mer and 21-mer formats are shown below in Table 4 .

TABLE 4. HEPCIDIN RNAi AGENTS 400 and 402 IN VARIOUS FORMATS

In the sequences in any of these tables or described elsewhere herein, any one or more U can be replaced by T, and these sequences can be represented by any nucleotide, e.g., ribonucleotides, or optionally one or more nucleotides can be modified or substituted.

[00237] For example, A106 modified variants of 400 and 402 successfully mediated RNA interference against Hepcidin in Huh7. In these A106 variants, in the anti-sense strand positions

3, 4, 13 and 15 of 400 and positions 3, 5, 6 and 15 of 402 are 2'-OMe; and positions 17 and 18 in 400 and 402 are 2'-MOE; in addition, the antisense strands both further comprise a spacer (ribitol), a phosphate and a 3' end cap (C6). These molecules demonstrated IC50 of less than 1 nM in Huh7 cells in vitro.

[00238] In other modified variants of 400 and 402, 1, 2 or 3 (or more) ribonucleotides at positions 4 to 8 of the antisense strand (in the so-called "seed region") can be individually replaced by DNA. See Fig. 3A and 3C and 4, for example. With both 400 and 402, these substitutions all yielded efficacious RNAi agents (e.g., with an IC50 in Huh7 cells of cell than 2 nM). In addition to being well-tolerated, DNA in the seed region also reduced the off-target phenotype (as measured by EC50 in HeLa cells). [00239] A variety of RNAi agents were constructed with the sequences of hs HAMP (human Hepcidin) 400 and 402, wherein the 3' terminus of the anti-sense strand further comprised, in 5' to 3' order, a spacer (e.g., ribitol), a phosphate, and a 3' end cap, e.g., BP, C6, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068 and X069. Other constructs were made using the 400 and 402 sequences, wherein the 3' terminus of the anti-sense strand further comprised a 3' end cap, e.g., BP, C6, X027, X038, X050, X051 , X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068 and X069. All of these constructs, which had a variety of stem chemistry modifications (with and without the 2'- MOE clamp, and with various 2'-OMe modifications), mediated RNA interference against Hepcidin, e.g., at least about 75-80% knockdown at 20 nM in Huh7 cells in vitro. [00240] A variety of RNAi agents were constructed with the hs HAMP (human Hepcidin) sequences designated either 400 and 402, wherein each sequence was further modified with a choice of chemical components as described by labels in Fig. 2A and 2B and in the figure legends. These modifications include: pattern of 2'OMe modifications in the stem sequence, presence or absence of ribitol spacer, and presence or absence of 2"methoxyethyl (MOE) clamp. One particular series of examples is shown in the graph, which depicts the activity of specific sequences with or without possible modifications. As shown, the 400 and 402 sequences differ at the 3' terminus of the anti-sense strand by chemical modifications comprising the PAZ ligand listed at the top of each graph segment, and further modifications, in 5' to 3' order, (i) at the 2' nucleotide comprising a stem sequence modification chosen from S92, S93, S38 and/or S42; (ii) a linkage between the final nucleotide of the sequence and the PAZ ligand selected from either a bond ("-") or a spacer (e.g., ribitol, shown as "Rib"); and (iii) the absence ("-") or presence ("MOE") of a 2'MOE clamp. [00241] Of the constructs shown, particular PAZ ligands (3' end caps) present at the 3' end of the anti-sense strand were selected from, but not limited to, e.g., BP, C6, X027, X038, X050, X051 , X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068 and X069. For the constructs of the invention, a variety of stem chemistry modifications are contemplated, wherein the pattern of 2'-OMe modifications to the stem portion of the nucleotide include, but are not limited to, S92, S93, S38 and/or S42. For each stem chemistry, further 2' nucleotide modifications may be present, e.g., constructs may be created with and without the 2'-MOE clamp. Therapeutically relevant constructs include those which mediate RNA interference against Hepcidin, e.g., at least about 75-80% knockdown at 20 nM in Huh7 cells in vitro.

[00242] 18-mer format [00243] The present disclosure contemplates a variety of different formats for Hepcidin RNAi agents, including various formats using any Hepcidin RNAi agent sequence disclosed herein. [00244] A particular novel format, designated the 18-mer format, has been developed. This and any other format can be used for a Hepcidin RNAi agent.

[00245] siRNAs naturally generated in a cell by Dicer typically comprise two 2 1-nt RNA strands, which form a 19-bp duplex region and two dinucleotide overhangs. This is the so- called "canonical" siRNA structure. See, for example, Elbashir et al. 2001 Nature 4 1 1: 494-498; Elbashir et al. 2001 EMBO J. 20: 6877. This is the so-called "canonical" structure or "21-mer" format of siRNAs. [00246] The two dinucleotide overhangs do not contribute to target specificity. Elbashir et al.

2001 Nature 4 1 1: 494-498; Elbashir et al. 2001 EMBO J. 20: 6877-6888; and Kraynack et al. 2006 RNA 12:163-176. They do, however, help protect the ends of the siRNA from nuclease degradation and sometimes improve activity. [00247] However, these di-nucleotides can optionally be replaced by 3' end caps. Ideal 3' end caps allow RNA interference activity, while increasing stability, e.g., against nucleases such as those in blood serum or intestinal fluid. U.S. Pat. Nos. 8,097,716; 8,084,600; 8,404,831 ; and 8,404,832. [00248] Novel 3' end caps (some described as "PAZ ligands") are disclosed herein; these can be used in place of or in addition to a 3' end cap in Hepcidin RNAi agents. It is noted that, although some 3' end caps are designated "PAZ ligands", this disclosure is not bound by any particular theory. [00249] The disclosure relates to compositions comprising a RNAi agent having a novel format (the "18-mer format"). These RNAi agents comprise a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand further comprises, in 5' to 3' order: a spacer; a phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, each strand terminates at the 5' end with a hydroxyl, optionally linked to a 5' end cap or a ligand, and terminates at the 3' end with a phosphate or modified internucleoside linker. In some embodiments, the 3' end of both the sense and anti-sense strand further comprise, in 5' to 3' order: a spacer; a phosphate or modified internucleoside linker; and a 3' end cap. The two strands can have the same or different spacers, phosphate or modified internucleoside linker, and/or 3' end caps. The strands can be ribonucleotides, or, optionally, one or more nucleotide can be modified or substituted. Optionally, at least one nucleotide comprises a modified internucleoside linker. Optionally, the RNAi agent can be modified on one or both 5' end. Optionally, the sense strand can comprise a 5' end cap which reduces the amount of the RNA interference mediated by this strand. Optionally, the RNAi agent is attached to a ligand. The disclosure also relates to processes for making such compositions, and methods and uses of such compositions, e.g., to mediate RNA interference.

[00250] In one embodiment, the 3' end of both the sense and anti-sense strand terminate in a phosphate or modified internucleoside linker and further comprise in 5' to 3' order: a spacer, a phosphate or modified internucleoside linker, and a 3' end cap.

[00251] In various embodiments, the spacer is ribitol or other type of abasic nucleotide, 2'- deoxy-ribitol, diribitol, 2'-methoxyethoxy-ribitol (ribitol with 2 -MOE), C3, C4, C5, C6, or 4- methoxybutane-1,3-diol (5300). In various embodiments, the spacer is not a nucleotide (which comprises a phosphate, a sugar and a base), as the spacer does not comprise all three elements; e.g., the spacer does not comprise a phosphate, a sugar and a base. In various embodiments, the spacers on the sense and anti-sense strands can be the same or different.

[00252] In various embodiments, the modified internucleoside linker is selected from phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide

3 - linker, and a compound of formula (I): (I), where R is selected from O - S , NH2,

BH3, CH3, C1-6 alkyl, C6-io aryl, C6-10alkoxy and C6-10 aryl-oxy, wherein C1-6 alkyl and C6-10 aryl are unsubstituted or optionally independently substituted with 1 to 3 groups independently selected

4 from halo, hydroxyl and NH2; and R is selected from O, S, NH, or CH2. [00253] In various embodiments, the 3' end cap is selected from those represented by formula 1a or 1b, disclosed in Tables 5A, 5B, 5C, 5D, 5E, or otherwise described herein. In various embodiments, the 3' end caps on the sense and anti-sense strands can be the same or different.

[00254] In one embodiment, the 3' end cap encompasses a compound of formula la: la

in which: X is a 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising: a strand of a Hepcidin RNAi agent, wherein the 3' end of the strand

terminates in a phosphate or modified internucleoside linker and optionally further comprises, in 5' to 3' order: a spacer and a second phosphate or modified internucleoside linker; Y is selected from CH and N;

m is selected from 0 and 1;

p is selected from 1, 2 and 3;

R3 is selected from hydrogen, 2-(hydroxy-methyl)-benzyl, 3-(hydroxy-methyl)-benzyl

and succinate; or is attached to a solid support; wherein the (CH 2) m-0-R 3 moiety is attached to the phenyl ring at position 3 or 4 ;

R4 is hydrogen;

R5 is hydrogen; or R4 and R5, together with the phenyl rings to which R4 and R5 are attached, form 6H-benzo[c]chromene.

[00255] In various embodiments, the 3' end cap encompasses a compound selected from Table 5A.

TABLE 5A. 44 in which: X is a 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising: a strand of a Hepcidin RNAi agent, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and optionally further comprises, in 5' to 3' order: a spacer and a second phosphate or modified internucleoside linker. In other words, a Hepcidin RNAi agent can comprise, as a non-limiting example: a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: an optional spacer and an optional second phosphate or modified internucleoside linker (wherein the strand, spacer, second phosphate or modified internucleoside linker are collectively "X), and a 3' end cap as shown herein. In another embodiment, a Hepcidin RNAi agent can comprise: a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker (wherein the strand is "X") and wherein the 3' end of the strand further comprises a 3' end cap as shown herein.

In one embodiment, the 3' end cap encompasses a compound of formula lb: lb in which: X is a 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising: a strand of a Hepcidin RNAi agent, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and optionally further comprises, in 5' to 3' order: a spacer and a second phosphate or modified internucleoside linker; q is selected from 0, 1 and 2;

R6 is selected from phenyl which is unsubstituted or substituted with a group selected from benzoxy and 3,4-dihydroxybutyl;

R7 is selected from hydrogen and hydroxy-ethyl, wherein if R is hydroxy-ethyl, the hydroxyl can be optionally functionalized as succinate or attached to a solid support;

R8 is selected from hydrogen and methoxy; Y is selected from CH and N; and

Y2 is selected from N and CR9; wherein R9 is selected from hydrogen and methyl.

various embodiments, the 3' end cap encompasses a compound selected from

in which: X is a 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising: a strand of a Hepcidin RNAi agent, wherein the 3' end of the strand

terminates in a phosphate or modified internucleoside linker and optionally further comprises, in 5' to 3' order: a spacer and a second phosphate or modified internucleoside linker. In other words, a Hepcidin RNAi agent can comprise, as a non-limiting example: a

strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: an optional spacer and an optional second phosphate or modified internucleoside linker (wherein the strand, spacer, second phosphate or modified internucleoside linker are collectively "X), and a 3' end cap as shown herein.

In another embodiment, a Hepcidin RNAi agent can comprise: a strand, wherein the 3'

end of the strand terminates in a phosphate or modified internucleoside linker (wherein the strand is "X") and wherein the 3' end of the strand further comprises a 3' end cap as shown herein.

[00258] In various embodiments, the 3' end cap encompasses a compound selected from Table 5C.

TABL in which: X is a 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising: a strand of a Hepcidin RNAi agent, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and optionally further comprises, in 5' to 3' order: a spacer and a second phosphate or modified internucleoside linker, and q is selected from 1 and 2 . In other words, a Hepcidin RNAi agent can comprise, as a non-limiting example: a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: an optional spacer and an optional second phosphate or modified internucleoside linker (wherein the strand, spacer, second phosphate or modified internucleoside linker are collectively "X), and a 3' end cap as shown herein. In another embodiment, a Hepcidin RNAi agent can comprise: a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker (wherein the strand is "X") and wherein the 3' end of the strand further comprises a 3' end cap as shown herein. various embodiments, the 3' end cap encompasses a compound selected from 53

X is a 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising: a strand of a Hepcidin RNAi agent, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and optionally further comprises, in 5' to 3' order: a spacer and a second phosphate or modified internucleoside linker. In other words, a Hepcidin RNAi agent can comprise, as a non-limiting example: a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: an optional spacer and an optional second phosphate or modified internucleoside linker (wherein the strand, spacer, second phosphate or modified internucleoside linker are collectively "X), and a 3' end cap as shown herein. In another embodiment, a Hepcidin RNAi agent can comprise: a strand, wherein the 3'

end of the strand terminates in a phosphate or modified internucleoside linker (wherein the strand is "X") and wherein the 3' end of the strand further comprises a 3' end cap as shown herein.

[00260] In various embodiments, the 3' end cap encompasses a compound selected from Table 5E.

TABLE 5E.

X is a 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising: a strand of a Hepcidin RNAi agent, wherein the 3' end of the strand

terminates in a phosphate or modified internucleoside linker and optionally further comprises, in 5' to 3' order: a spacer and a second phosphate or modified internucleoside linker. In other words, a Hepcidin RNAi agent can comprise, as a non-limiting example: a

strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: an optional spacer and an optional second phosphate or modified internucleoside linker (wherein the strand, spacer, second phosphate or modified internucleoside linker are collectively "X), and a 3' end cap as shown herein. In another embodiment, a Hepcidin RNAi agent can comprise: a strand, wherein the 3'

end of the strand terminates in a phosphate or modified internucleoside linker (wherein the strand is "X") and wherein the 3' end of the strand further comprises a 3' end cap as shown herein.

[00261] Table 5F illustrates specific 3' end caps, bound to the 3' terminal phosphate or modified internucleoside linker.

[00262] In the structures of Tables herein:

[00263] In some embodiments, hydroxyl groups are present and X represents the 3' end of a molecule comprising, in 5' to 3' order, an 18-mer strand of a RNAi agent, wherein the 3' end of the 18-mer strand terminates in a phosphate or modified internucleosider linker and further comprises, in 5' to 3' order: a spacer, and a phosphate or modified internucleoside linker. For example, the 3' end of a strand of a RNAi can terminate at a phosphate group, to which is bound (or further comprises), in 5' to 3' order: the spacer, the phosphate or modified internucleoside linker, and the 3' end cap is bound. Non-limiting examples of such a structure are shown in, for example, Fig. 15A (C3), Fig. 18C (C6, C8, and C10); and Fig. 19 (X027, C6 and X058). Table 5F shows the structures of various 3' end caps bound to the phosphate at the 3' end of a strand of an RNAi agent. [00264] A Hepcidin RNAi agent can comprise: a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: an optional spacer and an optional second phosphate or modified internucleoside linker (wherein the strand, spacer, second phosphate or modified internucleoside linker are collectively "X), and a 3' end cap as shown herein. [00265] In another embodiment, a Hepcidin RNAi agent can comprise: a strand, wherein the

3' end of the strand terminates in a phosphate or modified internucleoside linker (wherein the strand is "X") and wherein the 3' end of the strand further comprises a 3' end cap as shown herein.

[00266] In some embodiments, where hydroxyl groups in the 3' end caps are present, the hydroxyl can exist in a protected form. Suitable protecting groups for OH are known in the art. Protected forms of OH include, but are not limited to, ethers, phosphate esters, methyl tetraacetyl glucuronates, peracetyl glycosides and amino acid polypeptide esters. [00267] Synthesis schemes for producing various 3' end caps disclosed herein are provided in U.S. Provisional Patent Applications 61/886,739 and 61/886,748 and related applications. [00268]

[00269] Table 5F and 5F1 , below, presents some structures of various 3' end caps, including those suitable for RNAi-mediating molecules of sequences shown in Table 1. In several of the structures, the terminal 3' phosphate of a RNAi agent strand is also shown for context, although this phosphate is not part of the 3' end cap.

TABLE 5F. 3' end caps suitable for Hepcidin RNAi agents. X indicates the 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising a strand of Hepcidin RNAi agent which terminates in a phosphate or modified internucleoside linker; or, in 3' to 5' order: a phosphate or modified internucleoside linker; a spacer; and another phosphate or modified internucleoside linker.

TABLE 5F1. Hepcidin RNAi agents wherein the 3' end of a strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a phosphate, and a 3' end cap. The phosphate and 3' end cap are shown. 3 4

6

8 9

Additional structures include a phosphate conjugated to X1062, X1063 or X1064.

TABLE 5G. Hepcidin RNAi agents comprising a strand, wherein, as shown here, the 3' end of the strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer, a second phosphate, and a 3' end cap. The 3' terminal phosphate of the strand, spacer, second phosphate and 3' end cap are shown. 3 4 5 Additional structures include, inter alia: ribpX027, ribpX038, ribpX050, ribpX051, ribpX052, ribpX059, ribpX060, ribpX061 , ribpX062, ribpX063, ribpX064, ribpX065, ribpX066, ribpX067, ribpX068, ribpX069, ribpX097, ribpX098, ribpX109, ribpXHO, ribpX1 1, ribpX1 12, ribpX1 13, ribpX1009, ribpX101 1, ribpX1012, ribpX1013, ribpX1015, ribpX1016, ribpX1017, ribpX1018, ribpX1019, ribpX1020, ribpX1021 , ribpX1022, ribpX1024, ribpX1025, ribpX1026, ribpX1027, ribpX1028, ribpX1047, ribpX1048, ribpX1049, ribpX1062, ribpX1063, and ribpX1064. X1062, X1063 and X1064 are described herein and below:

These represent a spacer which is ribitol, a phosphate, and a 3' end cap which is X027, X038, X050, etc.

TABLE 5H. Hepcidin RNAi agents comprising a strand, wherein, as shown here, the 3' end of the 18-mer strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer, a modified internucleoside linker, and a 3' end cap. Example modified internucleoside linkers and 3' end caps are shown.

[00270] Regarding Tables 5F to 5H and other tables: [00271] Synthesis schemes for C7 amino and C3 amino (also designated amino C7 or amino C3, respectively) are not provided, as these molecules are commercially available from and synthesis schemes were previously published by Glen Research (Sterling, VA). [00272] C7 amino: Catalog Number: 20-2957-xx; Description: 3'-Amino-Modifier C7 CPG 500; 2-Dimethoxytrityloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-long chain alkylamino-CPG; Technical Bulletin: Pre-Synthesis Labeling of Aminomodifier C3 or C7 CPG, Glen Research (Sterling, VA). [00273] C3 amino: Catalog Number: 20-291 3-xx; Description: 3'-Spacer C3 CPG; (1- Dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPG, Glen Research (Sterling, VA). Glen Research also notes that Glen Research has no definitive data on the propyl CPG to support the assertion that it protects oligos from exonuclease digestion and does not permit polymerase extension. Glen Research's conclusion is based by analogy to the propylamino-modifier CPG [Zendegui et al. Nucleic Acids Research, 1992, 20, 307-314] (Cat. No. 20-2950-41). This modification protects oligos from exonuclease digestion but permits polymerase extension to a small extent since the modifier is eliminated to a level of about 10% from the 3' terminus, leaving the 3'-hydroxyl group available. HPLC experiments have shown that there is no detectable elimination of the propyl group from oligos made from the spacer C3- CPG. [00274] Synthesis schemes for producing various other 3' end caps disclosed herein are provided in U.S. Provisional Patent Applications 61/886,739 and 61/886,748 and related applications.

[00275] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X109, X 110, X 111, X 112, X 113, X1009, X1010, X101 1, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021 , X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062, X1063, X1064, or ribitol or other type of abasic nucleotide, or any 3' end cap disclosed herein or known in the art.

[00276] In various embodiments, the 3' end cap is C3, C6, C12, Triethylene glycol, Cyclohexyl (or Cyclohex), Phenyl, Biphenyl, Adamantane and Lithocholic acid (or Lithochol). These are described herein and/or in U.S. Pat. Nos. 8,097,716; 8,084,600; 8,344,128; 8,404,831 ; and 8,404,832.

[00277] In some embodiments, the 3' end cap is a ribitol. Thus, in some embodiments, the RNAi agent comprises an 18-mer strand further comprising at the 3' terminus, in 5' to 3' order, a spacer (e.g., a ribitol or other type of abasic nucleotide, C3, C4, C5, C6, etc.), a phosphate or modified internucleoside linker, and a 3' end cap (e.g., a second ribitol or other type of abasic nucleotide).

[00278] In some embodiments, the 3' end cap is a diribitol. Thus: In some embodiments, the RNAi agent comprises an 18-mer strand further comprising at the 3' terminus, in 5' to 3' order, a spacer (e.g., a ribitol, C3, C4, C5, C6, etc.), a phosphate or modified internucleoside linker, and a 3' end cap (e.g., a diribitol). [00279] Additional information can be found in U.S. patent applications 61/886,753; 61/930,681; 61/886,748; 61/886,739; and 61/886,760, which are all incorporated by reference in their entirety.

[00280] ADDITIONAL ASPECTS OF A HEPCIDIN RNAi AGENT IN A 18-MER FORMAT [00281] As noted above, the disclosure thus relates to compositions comprising a novel format, which can be used to devise Hepcidin RNAi agents. These RNAi agents comprise a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the 3' end of both the sense and anti-sense strand further comprise, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. The two strands can have the same or different spacers, phosphate or modified internucleoside linker, and/or 3' end caps. In various embodiments, one or more nt can be modified and/or substituted. Various spacers, modified internucleoside linkers and 3' end caps are described below.

[00282] SPACERS: RIBITOL, DIRIBITOL, 2'-DEOXYRIBITOL, 2'-METHOXYETHOXY RIBITOL, C3, C4, C5, C6, OR 4-METHOXYBUTANE-1,3-DIOL (5300) [00283] In the present disclosure, in a Hepcidin RNAi agent, any of various spacers can be used in combination with strands of any sequence, with or without substitutions and/or modifications of nt, with phosphates or modified nucleoside spacers, and with any 3' end cap, in any combination without limitation. [00284] A spacer is a chemical moiety intended or used to create or maintain a space (e.g., a proper or functional spacing) between two other chemical moieties; e.g., between two phosphates or modified internucleoside linkers. In various embodiments, the proper or functional spacing can approximate the size of a ribitol spacer, C3 spacer, C4 spacer, C5 spacer, C6 spacer, or any other spacer disclosed herein. In various embodiments, the spacer is a ribitol, diribitol, 2'-deoxyribitol, or 2'-methoxyethoxy ribitol (ribitol with 2'-MOE) or an equivalent abasic nucleotide known to one skilled in the art, or a lower alkyl or alkoxy group such as a C3,

C4, C5 or C6, or 4-methoxybutane-1,3-diol. In various embodiments, the spacer is not a nucleotide (which comprises a phosphate, a sugar and a base), as the spacer does not comprise all three elements; e.g., the spacer does not comprise a phosphate, a sugar and a base. Various embodiments are described in more detail below.

[00285] Ribitol spacer. [00286] In some embodiments, the spacer is ribitol. [00287] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand (comprising a 3' terminal phosphate or a modified internucleoside linker); a spacer which is ribitol; a phosphate or a modified internucleoside linker; and a 3' end cap (e.g., any 3' end cap described herein or otherwise known in the art). In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand comprising a 3' terminal phosphate; a spacer which is ribitol; a phosphate or a modified internucleoside linker; and a 3' end cap. [00 structure of a 3' terminal phosphate and ribitol spacer is shown here:

[00289] In some documents, the ribitol spacer is designated as N027 (C027, etc.). [00290] One embodiment is shown in Fig. 18, wherein the RNAi agent comprises, in 5' to 3' order: an 18-mer strand, wherein the 3' end of the 18-mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is ribitol, a phosphate, and a 3' end cap which is X058. This structure can be on any RNAi strand of any sequence or target. In addition, any 3' end cap disclosed herein can be used in place of X058. [00291] A related structure is shown in Fig. 17 ("ribitol with X058"), wherein the last nucleotide of the 18-mer strand is shown (and is a 2'-MOE), and the 3' end of the 18-mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is ribitol, a second phosphate, and a 3' end cap which is X058. [00292] Another embodiment is shown in Fig. 17 ("ribitol with C6 cap"), wherein the last nucleotide of the 18-mer strand is shown (and is a 2'-MOE), and the 3' end of the 18-mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is ribitol, a phosphate, and a 3' end cap which is C6. [00293] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a ribitol spacer, a phosphate, and a C6 3' end cap. This is diagrammed as ribpC6 (or ribC6) in Table 2 . [00294] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a ribitol spacer, a phosphate, and a BP 3' end cap. This is diagrammed as ribpBP (or ribBP) in Table 2 . [00295] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a ribitol spacer, a phosphate, and a C10 3' end cap. This is diagrammed as ribpCIO (or ribCIO) in Table 2 .

[00296] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X109, X 110, X 111, X 112, X 113, X1009, X1010, X101 1, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021 , X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062, X1063, X1064, or ribitol, or any 3' end cap disclosed herein or known in the art.

[00297] In some embodiments, the 3' end cap is a ribitol. Thus, the RNAi agent comprises an 18-mer strand, wherein the 3' end of the 18-mer strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer which is ribitol, a second phosphate or modified internucleoside linker, and a 3' end cap which is a second ribitol. In one embodiment, the 3' end of the 18-mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is ribitol, a second phosphate, and a 3' end cap which is a second ribitol. Such as structure is illustrated in Fig. 17 (including the 3' terminal nucleotide and phosphate) and designated "diribitol". [00298] The structure comprising an RNAi agent comprising, in 5' to 3' order, an 18-mer strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a ribitol spacer, a phosphate or a modified internucleoside linker, and a 3' end cap (e.g., any 3' end cap disclosed herein) can be used on any RNAi agent of sequence or target, including but not limited to a double-stranded RNA, wherein optionally one or more phosphates are replaced by a modified internucleoside linker, optionally one or more nucleotides are modified, and optionally one or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, FANA, ANA, HNA, CeNA, and/or UNA.

[00299] Diribitol spacer. [00300] In some embodiments the spacer is Diribitol. [00301] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises in 5' to 3' order: a spacer (wherein the spacer comprises in 5' to 3' order: a first ribitol; a phosphate or a modified internucleoside linker; a second ribitol; and a phosphate or a modified internucleoside linker); and a 3' end cap.

[00302] Thus: In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand comprising a 3' terminal phosphate; a first ribitol; a phosphate; a second ribitol; a phosphate or a modified internucleoside linker; and a 3' end cap. [00303] This structure of a 3' terminal phosphate, a first ribitol, a phosphate, and a second ribitol is shown here:

[00304] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand comprising a 3' terminal phosphate; a first ribitol spacer; a phosphate; a second ribitol spacer; a phosphate or a modified internucleoside linker; and a 3' end cap. [00305] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a first ribitol spacer, a phosphate or a modified internucleoside linker, a second ribitol spacer, a phosphate or a modified internucleoside linker, and a 3' end cap which is a ribitol; this structure is designated a tri ribitol.

[00306] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X109, X 110, X 111, X 112, X 113, X1009, X1010, X101 1, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021 , X1022, X1024, X1025, X1026,

X 1027, X 1028, X 1047, X 1048, X 1049, X 1062, X 1063, or X 1064, or any 3' end cap disclosed herein or known in the art or known in the art. [00307] The structure comprising an RNAi agent comprising, in 5' to 3' order, an 18-mer strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a first ribitol spacer, a phosphate or a modified internucleoside linker, a second ribitol spacer, a phosphate or a modified internucleoside linker, and a 3' end cap (e.g., any 3' end cap disclosed herein) can be used on any RNAi agent of sequence or target, including but not limited to a double-stranded RNA, wherein optionally one or more phosphates are replaced by a modified internucleoside linker, optionally one or more nucleotides are modified, and optionally one or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA, HNA, FANA, CeNA, and/or UNA.

[00308] 2'-methoxyethoxy ribitol spacer. [00309] In some embodiments, the spacer is 2'-methoxyethoxy ribitol or other type of abasic nucleotide.

[00310] In one embodiment, the RNAi agent comprises a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer which is 2'-methoxyethoxy ribitol, a second phosphate or modified internucleoside linker, and a 3' end (e.g., any 3' end cap described herein or known in the art).

In other words: In one embodiment, the RNAi agent comprises, in 5' to 3' order: a strand comprising a 3' terminal phosphate or a modified internucleoside linker; a spacer which is 2'- methoxyethoxy ribitol; a phosphate or a modified internucleoside linker; and a 3' end cap (e.g., any 3' end cap described herein or known in the art). Thus: In one embodiment, the RNAi agent comprises, in 5' to 3' order: a strand comprising a 3' terminal phosphate; a spacer which is 2'- methoxyethoxy ribitol; a phosphate or a modified internucleoside linker; and a 3' end cap. [00311] The structure of the 3' terminal phosphate and 2'-methoxyethoxy ribitol spacer is

shown here: 2'-methoxyethoxy ribitol spacer. [00312] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand, wherein the 3' end of the 18-mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is 2'-methoxyethoxy ribitol, a phosphate, and a 3' end cap which is

X058. This structure can be on any RNAi strand of any sequence or target. In addition, any 3' end cap disclosed herein can be used in place of X058. [00313] A related structure is 2'-methoxyethoxy ribitol with X058, wherein the last nucleotide of the 18-mer strand is a 2'-MOE), and the 3' end of the 18-mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is 2'-methoxyethoxy ribitol, a second phosphate, and a 3' end cap which is X058. [00314] Another embodiment is 2'-methoxyethoxy ribitol with C6 cap, wherein the last nucleotide of the 18-mer strand is a 2'-MOE), and the 3' end of the 18-mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is 2'-methoxyethoxy ribitol, a phosphate, and a 3' end cap which is C6. [00315] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a 2'-methoxyethoxy ribitol spacer, a phosphate, and a C6 3' end cap. [00316] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a 2'-methoxyethoxy ribitol spacer, a phosphate, and a BP 3' end cap. [00317] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a 2'-methoxyethoxy ribitol spacer, a phosphate, and a C10 3' end cap.

[00318] In another embodiment, the RNAi agent comprises a strand, wherein the 3' end of the strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is 2'-methoxyethoxy ribitol, a phosphate, and a 3' end cap which is X058.

[00319] In some embodiments, the 3' end cap is a 2'-methoxyethoxy ribitol. Thus, the RNAi agent comprises an 18-mer strand, wherein the 3' end of the 18-mer strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer which is 2'-methoxyethoxy ribitol, a second phosphate or modified internucleoside linker, and a

3' end cap which is a second 2'-methoxyethoxy ribitol. In one embodiment, the 3' end of the 18- mer strand terminates in a phosphate and further comprises, in 5' to 3' order: a spacer which is 2'-methoxyethoxy ribitol, a second phosphate, and a 3' end cap which is a second 2'- methoxyethoxy ribitol.

[00320] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X109, X 110, X 111, X 112, X 113, X1009, X1010, X101 1, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021 , X1022, X1024, X1025, X1026, X1027, X1028, X1047, X1048, X1049, X1062, X1063, X1064, or 2'-methoxyethoxy ribitol, or any 3' end cap disclosed herein or known in the art. The structure comprising an RNAi agent comprising, in 5' to 3' order, a strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a 2'-methoxyethoxy ribitol spacer, a phosphate or a modified internucleoside linker, and a 3' end cap (e.g., any 3' end cap disclosed herein including but not limited to those listed in the previous sentence) can be used on any RNAi agent of any length, sequence or target, including but not limited to a double-stranded RNA, wherein optionally one or more phosphates are replaced by a modified internucleoside linker, optionally one or more nucleotides are modified, and optionally one or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

[00321] 2'-deoxyribitol spacer. [00322] In some embodiments the spacer is 2'-deoxyribitol. [00323] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate or a modified internucleoside linker, a spacer which is 2'- deoxyribitol (2'-deoxyrib), a phosphate or a modified internucleoside linker, and a 3' end cap. [00324] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a spacer which is 2'-deoxyribitol (2'-deoxyrib), a phosphate or a modified internucleoside linker, and a 3' end cap. The structure of a 3' terminal phosphate and

a 2'-deoxyribitol is shown here : [00325] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a 2'-deoxyribitol spacer, a phosphate, and a C12 3' end cap. This is diagrammed as ribpCIO (or ribCIO) in Table 2 . This embodiment is designated "2'DeoxyribC12" and illustrated in Table 2 .

[00326] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X109, X 110, X 111, X 112, X 113, X1009, X1010, X101 1, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021 , X1022, X1024, X1025, X1026, X 1027, X 1028, X 1047, X 1048, X 1049, X 1062, X 1063, or X 1064, or any 3' end cap disclosed herein or known in the art.

[00327] The structure comprising an RNAi agent comprising, in 5' to 3' order, an 18-mer strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a 2'- deoxyribitol spacer, a phosphate or a modified internucleoside linker, and a 3' end cap (e.g., any 3' end cap disclosed herein) can be used on any RNAi agent of any sequence or target, including but not limited to a double-stranded RNA, wherein optionally one or more phosphates are replaced by a modified internucleoside linker, optionally one or more nucleotides are modified, and optionally one or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

[00328] C3 spacer. [00329] In various embodiments, the spacer is C3.

[00330] In one embodiment, the RNAi agent comprises an 18-mer strand, wherein the 3' end of the 18-mer strand terminates in a 3' phosphate or a modified internucleoside linker, and further comprises a spacer which is C3, a phosphate or a modified internucleoside linker, and a 3' end cap.

[00331] In one embodiment, the RNAi agent comprises two 18-mer strands, wherein the 3' end of each 18-mer strand terminates in a 3' phosphate or a modified internucleoside linker, and further comprises a spacer, a second phosphate or a modified internucleoside linker, and a 3' end cap, wherein the spacer in one or both strands is C3.

[00332] The C3 spacer has the chemical formula -(CH 2 )3-. The structure of a 3' terminal

[00333] One embodiment is shown in Fig. 18, wherein the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a C3 spacer, a phosphate, and a 3' end cap which is X058. This structure can be on any RNAi strand of any sequence or target. In addition, any 3' end cap disclosed herein or known in the art can be used in place of X058, and any modified internucleoside linker can be used in place of phosphate. [00334] Another embodiment is shown in Fig. 11, which illustrates a portion of a RNAi agent to Factor VII comprising an 18-mer strand, wherein the 18-mer strand terminates in a phosphate and further comprises in 5' to 3' order: a C3 spacer, a phosphate and a 3' end cap which is C6. This is designated "C3pC6 overhang". This structure can be on any RNAi strand of any sequence or target. In addition, any 3' end cap disclosed herein or known in the art can be used in place of C6, and any modified internucleoside linker can be used in place of phosphate. [00335] The efficacy of a RNAi agent comprising a C3 spacer is shown in Fig. 19. Two different HuR constructs were prepared comprising an 18-mer, wherein the 3' end of the 18-mer terminates in a phosphate and further comprises in 5' to 3' order: a C3 spacer, a phosphate and a 3' end cap (which is C6 or X058). Both of these were able to mediate RNA interference.

[00336] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X109, X 110, X 111, X 112, X 113, X1009, X1010, X101 1, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021 , X1022, X1024, X1025, X1026,

X 1027, X 1028, X 1047, X 1048, X 1049, X 1062, X 1063, or X 1064, or any 3' end cap disclosed herein or known in the art. [00337] The structure comprising an RNAi agent comprising, in 5' to 3' order, an 18-mer strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a C3 spacer, a phosphate or a modified internucleoside linker, and a 3' end cap (e.g., any 3' end cap disclosed herein) can be used on any RNAi agent of any sequence or target, including but not limited to a double-stranded RNA, wherein optionally one or more phosphates are replaced by a modified internucleoside linker, optionally one or more nucleotides are modified, and optionally one or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

[00338] C4 spacer, C5 spacer and C6 spacer.

[00339] In various embodiments, the spacer is C4 or C5 or C6. [00340] In one embodiment, the Hepcidin RNAi agent comprises an 18-mer strand, wherein the 3' end of the 18-mer strand terminates in a 3' phosphate or a modified internucleoside linker, and further comprises a spacer which is C4 or C5 or C6, a phosphate or a modified internucleoside linker, and a 3' end cap. [00341] In one embodiment, the RNAi agent comprises two 18-mer strands, wherein the 3' end of each 18-mer strand terminates in a 3' phosphate or a modified internucleoside linker, and further comprises a spacer, a second phosphate or a modified internucleoside linker, and a 3' end cap, wherein the spacer in one or both strands is C4 or C5 or C6. [00342] The C3 to C6 spacers can be defined as: C3 = 1,3-propane-diol C4 = 1,4-butane-diol C5 = 1,5-pentane-diol C6 = 1,6-hexane-diol [00343] In some contexts:

[00344] The C4 spacer has the chemical formula -(CH 2 )4-.

[00345] The C5 spacer has the chemical formula -(CH 2)5-.

[00346] The C6 spacer has the chemical formula -(CH 2)6- . [00347] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP

(biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C 10, C12, X027, X038, X050, X05 1, X052, X058, X059, X060, X06 1, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X 109, X 110, X 111, X 112, X 113, X 1009, X 1010, X 1011, X 1012 ,

X 10 13, X 10 15, X 10 16, X 10 17, X 1018, X 1019, X 1020, X 1021 , X 1022, X 1024, X 1025, X 1026,

X 1027, X 1028, X 1047, X 1048, X 1049, X 1062, X 1063, or X 1064, or any 3' end cap disclosed herein or known in the art.

[00348] The structure comprising an RNAi agent comprising, in 5' to 3' order, an 18-mer strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a C4 or C5 or C6 spacer, a phosphate or a modified internucleoside linker, and a 3' end cap (e.g., any 3' end cap disclosed herein) can be used on any RNAi agent of any sequence or target, including but not limited to a double-stranded RNA, wherein optionally one or more phosphates are replaced by a modified internucleoside linker, optionally one or more nucleotides are modified, and optionally one or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

[00349] As a note of clarification, this disclosure notes that the terms "C3" [-(CH 2)3-], "C4" [-

(CH2)4-], and "C5" [-(CH 2)5-] are generally used herein to designate spacers, similar terms (C3, C4, C5 "linkers") are also used to designate a portion of a 3' end cap, as illustrated in Fig. 20A, 20B and 20C. In these figures, the different linkers are used to differentiate portions various 3' end caps. It is also noted that the term "C3" is used to designate a C3 3' end cap (see, e.g, U.S. Pat. No. 8,097,716), a C3 spacer (Fig. 18), and a C3 linker (Fig. 26). The C6 spacer should also be differentiated from the C6 end cap.

[00350] 4-methoxybutane-1,3-diol (5300) spacer. [00351] In various embodiments, the spacer is 4-methoxybutane-1,3-diol. 4-methoxybutane- 1,3-diol is also designated 5300, A5300, C5300, G5300, and UG5300.

[00352] In one embodiment, the Hepcidin RNAi agent comprises, in 5' to 3' order: an 18-mer strand, wherein the 3' end terminates in a 3' phosphate (a 3' terminal phosphate) or a modified internucleoside linker and further comprises: a spacer which is 4-methoxybutane-1 ,3-diol, a phosphate or a modified internucleoside linker, and a 3' end cap. [00353] The structure of a 3' terminal phosphate and a 4-methoxybutane-1,3-diol spacer is

shown here: [00354] In one embodiment, the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a spacer which is 4-methoxybutane-1 ,3-diol, a phosphate or a modified internucleoside linker, and a 3' end cap. [00355] One embodiment is shown in Fig. 18, wherein the RNAi agent comprises, in 5' to 3' order: an 18-mer strand terminating in a 3' phosphate, a 4-methoxybutane-1,3-diol spacer, a phosphate, and a 3' end cap which is X058. This structure can be on any RNAi strand of any sequence or target. In addition, any 3' end cap disclosed herein or known in the art can be used in place of X058, and any modified internucleoside linker can be used in place of phosphate. [00356] The efficacy of a RNAi agent comprising a C5300 spacer is shown in Fig. 19. Two different HuR constructs were prepared comprising an 18-mer, wherein the 3' end of the 18-mer terminates in a phosphate and further comprises a C5300 spacer, a phosphate and a 3' end cap (which is C6 or X058). Both of these were able to mediate RNA interference. [00357] In various embodiments, the 3' end cap is triethylene glycol, cyclohexyl, phenyl, BP (biphenyl), lithochol (lithocholic acid), adamantane, C3 amino, C7 amino, C3, C6, C8, C10, C12, X027, X038, X050, X051, X052, X058, X059, X060, X061, X062, X063, X064, X065, X066,

X067, X068, X069, X097, X098, X109, X 110, X 111, X 112, X 113, X1009, X1010, X101 1, X1012, X1013, X1015, X1016, X1017, X1018, X1019, X1020, X1021 , X1022, X1024, X1025, X1026,

X 1027, X 1028, X 1047, X 1048, X 1049, X 1062, X 1063, or X 1064, or any 3' end cap disclosed herein or known in the art. The structure comprising an RNAi agent comprising, in 5' to 3' order, an 18-mer strand terminating in a 3' terminal phosphate or a modified internucleoside linker, a 4-methoxybutane- 1,3-diol spacer, a phosphate or a modified internucleoside linker, and a 3' end cap (e.g., any 3' end cap disclosed herein) can be used on any RNAi agent of any sequence or target, including but not limited to a double-stranded RNA, wherein optionally one or more phosphates are replaced by a modified internucleoside linker, optionally one or more nucleotides are modified, and optionally one or more RNA nucleotides are replaced by DNA, PNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA.

[00358] PHOSPHATE OR MODIFIED INTERNUCLEOSIDE LINKER

[00359] In various embodiments, the modified internucleoside linker is: phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker, or a compound of formula (I), as detailed below. [00360] The disclosure relates to compositions comprising a Hepcidin RNAi agent having a novel format. These RNAi agents comprise a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. In some embodiments, the 3' end of both the sense and anti-sense strand further comprise, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap. The two strands can have the same or different spacers, phosphate or modified internucleoside linker, and/or 3' end caps. In various embodiments, one or more nt can be modified and/or substituted. Various spacers are described below. Various phosphates or modified internucleoside linkers can be used in combination with strands of any sequence, with or without substitutions and/or modifications of nt, with any spacers, and with any 3' end cap, in any combination without limitation. [00361] In some embodiments, the modified internucleoside linker is interposed between the spacer and the 3' end cap.

[00362] In various embodiments, one or more of the phosphates of one or both strands of the RNAi agent are replaced. Thus: In various embodiments, one or more nucleotide of one or both strands has a modified internucleoside linker. In some embodiments, the 3' terminal phosphate is replaced. In some embodiments, one or more nucleotide of one or both strands has a modified internucleoside linker, and/or a modified internucleoside linker is interposed between the spacer and the 3' end cap.

[00363] In one embodiment, the present disclosure encompasses a RNAi agent comprising a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap, wherein the 3' end cap is selected from the 3' end caps listed in any Table herein or otherwise disclosed herein, and wherein at least one nucleotide has a modified internucleoside linker a modified internucleoside linker (e.g., wherein at least one phosphate of a nucleotide is replaced by a modified

internucleoside linker), where the modified internucleoside linker is: phosphorothioate

(PS), phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker,

or a compound of formula (I): (I), where R is selected from O , S , NH2, BH3, CH3, Ci_6 i o C _ alkyl, C6-ioaryl, C -6 alkoxy and C6- aryl-oxy, wherein 6 alkyl and C6-ioaryl are unsubstituted or optionally independently substituted with 1 to 3 groups independently selected 4 from halo, hydroxyl and NH2; and R is selected from O, S, NH, or CH2. [00364] In one embodiment, the present disclosure encompasses a RNAi agent comprising a sense and an anti-sense strand, each strand being an 18-mer and the strands together forming a blunt-ended duplex, wherein the 3' end of at least one strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer; a second phosphate or modified internucleoside linker; and a 3' end cap, wherein the 3' end cap is selected from the 3' end caps listed in any Table herein or otherwise disclosed herein, and wherein at least the 3' terminal nucleotide on one or both strands has a modified internucleoside linker (e.g., wherein the phosphate of the 3' nucleotide on one or both strands is replaced by a modified internucleoside linker), wherein the modified internucleoside linker is phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker, or a compound of formula (I).

[00365] In various embodiments, the 3' end cap is linked via a terminal phosphate group (i.e., a phosphate group at the 3' end of a RNAi agent strand). Such compounds are shown in, for example, Table 2 . Alternatively, in 3' to 5' order, a 3' end cap can be bound to a phosphate or modified internucleoside linker, which is bound to a spacer, which is bound to a phosphate or modified internucleoside linker bound to the 3' carbon at the 3' end of at least one RNAi agent strand.

[00366] In one embodiment, compounds of table 2 have a terminal phosphorothioate group bound to the 3' carbon at the 3' end of at least one RNAi agent strand. Thus, in various embodiments, in the 3' end caps listed in Table 2, the phosphate group is replaced by a phosphorothioate. In other words, the composition comprises a RNAi agent comprising a strand, wherein the 3' end of the strand terminates in a phosphorothioate and further comprises a compound of Table 2 (or any other 3' end cap described herein or known in the art). In additional embodiments, the phosphate group of various 3' end caps listed herein as C3, C6, C12, Triethylene glycol, Cyclohexyl, Phenyl, Biphenyl, Adamantane, Lithocholic acid can be replaced by phosphorothioate. In one particular embodiment, the phosphate group in the C3 3' end cap is replaced by phosphorothioate (and designated "PS-C3", as illustrated in Table 2 and described in Example 6 and FIGs. 20 A-E). In one particular embodiment, the phosphate group in the C6 3' end cap is replaced by phosphorothioate (and designated "PS-C6", as illustrated in

Table 2). In one particular embodiment, the phosphate group in the C10 3' end cap is replaced by phosphorothioate (and designated "PS-C10", as illustrated in Table 2). In one particular embodiment, the phosphate group in the biphenyl (BP) 3' end cap is replaced by phosphorothioate (and designated "PS-BP", as illustrated in Table 2). [00367] In various embodiments, R 1 = OH; and R2 = a compound of formula (I).

[00368] Various additional embodiments of Hepcidin RNAi agents in the 18-mer format

[00369] In some embodiments, the Hepcidin RNAi agent comprises a 18-mer strand further comprising at the 3' terminus a 3' end cap (but no spacer, or phosphate or internucleoside linker). Thus: In some embodiments, the RNAi agent comprises an 18-mer strand further comprising at the 3' terminus a 3' end cap (e.g., BP or C6).

[00370] In some embodiments, the Hepcidin RNAi agent comprises an 18-mer strand further comprising at the 3' terminus a spacer (but no phosphate or internucleoside linker, or 3' end cap). Thus: In some embodiments, the RNAi agent comprises an 18-mer strand further comprising at the 3' terminus a spacer (e.g., ribitol).

[00371] In various embodiments, one or both strands can comprise ribonucleotide subunits, or one or more nucleotide can optionally be modified or substituted. Thus, in various embodiments, the RNAi agent can either contain only naturally-occurring ribonucleotide subunits, or one or more modifications to the sugar, phosphate or base of one or more of nucleotide subunits. In one embodiment, the modifications improve efficacy, stability and/or reduce immunogenicity of the RNAi agent. [00372] One aspect of the present disclosure relates to a Hepcidin RNAi agent comprising at least one non-natural nucleobase. In certain embodiments, the non-natural nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a particular embodiment, the non- natural nucleobase is difluorotolyl. In certain embodiments, only one of the two strands contains a non-natural nucleobase. In certain embodiments, both of the strands contain a non-natural nucleobase.

[00373] In one embodiment, the first two base-pairing nucleotides on the 3' end of the sense and/or anti-sense strand are modified. In one embodiment, the first two base-pairing nucleotides on the 3' end of the sense and/or anti-sense strand are 2'-MOE (a 2' MOE clamp).

[00374] In one embodiment, the 3' terminal phosphate of the sense and/or anti-sense strands is replaced by a modified internucleoside linker.

[00375] In various embodiments, one or more nucleotides is modified or is substituted with DNA, a peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), or unlocked nucleic acid (UNA). In some embodiments, the replacement or substitution of RNA with DNA, or a nucleotide of a different backbone, or PNA, LNA, Morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA can be considered a "modification".

[00376] In various embodiments, at least one nucleotide comprises a modified internucleoside linker, wherein the modified internucleoside linker is selected from phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker, and a compound of formula (I).

[00377] In various embodiments, optionally the 3' terminal phosphate of the sense and/or anti-sense strands is replaced by a modified internucleoside linker.

[00378] In various embodiments, the RNAi agent can be modified on one or both 5' end. In various embodiments, the sense strand can comprise a 5' end cap which reduces the amount of the RNA interference mediated by this strand.

[00379] In various embodiments, the sense strand comprises a 5' end cap selected: a nucleotide lacking a 5' phosphate or 5'-OH; a nucleotide lacking a 5' phosphate or a 5'-OH and also comprising a 2-OMe or 2'-MOE modification; 5'-deoxy-2'-0-methyl modification; 5'-OME- dT; ddT; and 5'-OTr-dT.

[00380] In various embodiments, the RNAi agent is optionally attached to a ligand. The ligand can be selected to improve one or more characteristic, such as, e.g., stability, distribution and/or cellular uptake of the agent, e.g., cholesterol or a derivative thereof.

[00381] In various embodiments, the RNAi agent can be isolated or be part of a pharmaceutical composition used for the methods described herein.

[00382] In various embodiments, the pharmaceutical composition can be a lipid nanoparticle.

[00383] In various embodiments, the pharmaceutical composition can be a lipid nanoparticle Optionally, the pharmaceutical compositions can further comprise or be used in conjunction with any known treatment for any target gene-related disease. [00384] The present disclosure further provides methods for reducing the level of target gene mRNA in a cell, particularly in the case of a disease characterized by over-expression, over-abundance or hyper-activity of the target gene product. The present disclosure also encompasses a method of treating a human subject having a pathological state mediated at least in part by target gene expression, the method comprising the step of administering to the subject a therapeutically effective amount of a RNAi agent target gene. Such methods comprise the step of administering one of the RNAi agents of the present disclosure to a subject. Reduction of target gene mRNA in a cell results in a reduction in the amount of encoded target gene protein produced. [00385] In another embodiment, the invention provides an RNAi agent with any one or more of the above properties for use as a medicament. [00386] The methods and compositions of the present disclosure, e.g., the methods and target gene RNAi agent compositions, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

[00387] In various embodiments, the RNAi agent can be combined with one or more additional RNAi agents in the same formulation. The one or more additional RNAi agents can have the same or different sequences, targets, spacers, 3' end caps, nucleotide replacements modifications, and/or ligands, etc. In various embodiments, the one or more additional RNAi agents can have a sense and an anti-sense strand wherein each is an 18-mer and together form a blunt-ended duplex. The one or more additional RNAi agent can target the same or different sequence and/or the same or different target gene. [00388] Thus: Multiple RNAi agents can be administered separately or co-administered. The multiple RNAi agents can be administered in the same delivery vehicle, the same type of delivery vehicle, or in different delivery vehicles. [00389] Various additional embodiments are described below. [00390]

[00391] Use of various 3' end caps (e.g., ligands or PAZ ligands) with Hepcidin RNAi agents [00392] Any of the various 3' end caps (e.g., ligands or PAZ ligands) can be used with the Hepcidin RNAi agents described herein. [00393] A 3' end cap is a non-nucleotidic chemical moiety bound to the 3' end of a molecule comprising a RNAi agent, e.g., the 3' terminus (or 3' end) of (a) a molecule comprising a strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker; or (b) a molecule comprising, in 5' to 3' order: a strand (wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker), a spacer, and a second phosphate or modified internucleoside linker. The 3' end cap performs at least one of the following functions: allowing RNA interference mediated by the molecule, protecting the molecule from degradation or reducing the amount or rate of degradation of the molecule (e.g., by nucleases), reducing the off-target effects of the sense strand, or increasing the activity, duration or efficacy of RNA interference mediated by the molecule. By describing a 3' end cap as "non-nucleotidic", it is meant that a nucleotide comprises three components: a phosphate, a pentose (e.g., a ribose or deoxyribose) and a nucleobase, and a 3' end cap does not comprise all three of the components. [00394] 3' end caps include, but are not limited to compounds designated "PAZ ligands". These ligands can provide increased potency and/or higher siRNA turnover. [00395] Various PAZ ligands and 3' end caps have been tested with various Hepcidin RNAi agents. These include PAZ ligands: X027, X038, X050, X051 , X052, X058, X059, X060, X061 , X062, X063, X064, X065, X066, X067, X068, X069, X097, and X098, plus the 3' end caps designated C6 and BP (biphenol) in U.S. Patent No. 8,404,831 and 8,084,600, respectively. In several experiments, C6 is used as a positive control 3' end cap. It is noted that some documents also refer to C6, C8, C10, C12, BP and other 3' end caps as PAZ ligands. [00396] In one study, anti-sense strand of the mouse Hepcidin siRNA is conjugated to each of various 3' end caps (X052, X058, X067, X038, X069, X027, and C6 as a control) and the RNAi agent tested in vivo. At day 2, all RNAi agents mediated knockdown (at least about 50% knockdown compared to PBS control), with X058 having superior knockdown than C6 control.

At day 7, only the RNAi with X058 is still active in vivo (even the C6 control is no longer active). [00397] In an addition in vivo study, the X058 3' end cap again proved to be equal or more potent than the C6 3' end cap on Hepcidin RNAi agent in vivo at Day 2 . The 18-mer Hepcidin RNAi agent with the X058 also showed duration of activity equal to or better than the longer 21- mer Hepcidin RNAi agent. Thus, in at least some cases, the 18-mer format can be more potent than the 21-mer format. [00398] Two human Hepcidin RNAi agents (designated 400 and 402) were constructed, with the guide strand, with or without (+ or -) the MOE clamp (wherein the first two nt on the 3' end of each strand are 2'-MOE), with or without (+ or -) the ribitol spacer, and with each of the 17 listed PAZ ligands, C6 and BP as the 3' end caps. The passenger strands were constructed with or without (+ or -) the MOE clamp, with or without (+ or -) the ribitol spacer, and with C6 as the 3' end cap.

[00399] The RNAi agents are tested in a 3 point dose response. [00400] RNAi agents are introduced into Huh7 cells (10,000) using Optifect. [00401] Three RNAi agent concentrations are used: 10, 10 and 5 nM. [00402] A total of 152 siRNA agents are tested. [00403] 7x96-well plates are transfected in duplicate (with randomized distribution of siRNAs across the plates). [00404] However, 400 and 402 C6 are on every plate. [00405] qRT-PCR is performed and duplicate, and HAMP is normalized to GAPDH mRNA. [00406] Plate-to-plate normalization is also performed. [00407] Results are shown in Figs. 26 and 27. [00408] Trends include: For the 400 sequence, MOE clamp in combination with the ribitol spacer is the most active format. The wild-type and no ribitol spacer are least active. For the 402 sequence, the MOE clamp and ribitol spacer are the most active; wild-type and no ribitol spacer are least active.

[00409] At least five PAZ ligands consistently are among the most active at all doses: X038, X067, X069, X058 and X052, using four different chemical scaffolds. [00410] Modification of Human Hepcidin RNAi agents 400 and 402 [00411] Once efficacious siRNA sequences are identified, these siRNAs can be optimized by performing various modifications and testing those modifications to find those which improve efficacy, duration and/or stability and/or reduce off-target effects, or otherwise improve the performance of the siRNA. [00412] The efficacious human Hepcidin RNAi agents with sequences 400 and 402 can be modified by replacing a RNA with 2'-OMe-RNA, DNA, 2 -F-RNA, 2'-MOE-RNA, and/or addition of a spacer (e.g., ribitol), and/or additional of a 3' end cap (X058, C6 or other 3' end cap). [00413] Human hepcidin RNAi agents were thus modified in numerous ways and tested for activity. [00414] Human Hepcidin RNAi Agents with sequences 400 and 402 were also modified by replacement of RNA with DNA. In particular, nucleotides in the seed region can be replaced by DNA (e.g., U with T at position 4 , C at position 5, C at position 6, A at position 7, and A at position 8 of 400; and A at position 4, U with T at position 5, U with T at position 6, C at position 7 and C at position 8 of 402). RNAi names sometimes have suffices identifying a base as DNA (e.g., "pos4_DNA" indicates that the nucleotide at position 4 is DNA; "pos8_DNA" indicates that that at position 8 is DNA, etc.). These modifications reduce off-target effects by weakening the binding affinity of the guide strand seed region. Off-target effects can be measured, for example, as EC50 in nM in HeLa cells. The data show that DNA is thus well-tolerated, e.g., in the seed region and can reduce the off-target phenotype. In some cases, other positions (e.g., position 2 of 402) can be replaced by DNA, or more than one nucleotide can be replaced by DNA (e.g., positions 2 and 4 of 402). See Fig. 4, for example. [00415] Modifications can be made to increase the serum stability of 400 and 402. These siRNAs have been shown to be susceptible to cleavage after (3' to) the 5' terminal U (position

1), C (position 6) and C (position 11) (400); and after (3' to) the 5' terminal U (position 1), C (position 8), and C (position 13) (402). Modifications can thus also reduce this cleavage. [00416] Example modifications are shown herein.

[00417] In various experiments, off-target effects are measured in HeLa cells (1000 cells, using HiPerFect, 166 hours post-transfection); and potency is measured (e.g., 12 dose- response, time course, in Huh7 cells, using 8000 cells, using OptiFect, at 24, 48, 72, 120 and 166 hours). Potency reporter: human/cyno (6 dose-response): COS1 (5000 cells), Fugene/Oligofectamine, 72 hours; HAMP antisense reporter human, HAMP sense reporter human, and HAMP antisense reporter cyno. [00418] A portion of the data generated with these modifications and experimental conditions is shown in below, in TABLE 6.

[00419] TABLE 6. Properties of various modified variants of human Hepcidin RNAi agents 400 and 402. [00420] Col. (Column) 2 ("Huh7 kd") provides the residual mRNA relative to control; thus, in the first line, "15" indicates 15% residual activity, or 85% gene knockdown by the RNAi agent. Col. 4 similarly expresses knockdown by showing residual gene activity at 166 hours (approximately 7 days). All of the tested siRNAs showed significant knockdown of gene activity at 48 hours. In these experiments, particularly effective human Hepcidin RNAi agents with sequences 400 and 402 include those of designated "hs_HAMP_400_A107 *_3'X058_pos4_DNA_S42", which displays 13% residual gene activity at 166 hours; "hs_HAMP_402_A107 *_3'X058_pos4_DNA_S42", and "hs_HAMP_A107 *_3'X058_pos2,4_DNA_3'X058_S42". Of these modified siRNAs, "hs_HAMP_400_A1 07*_3'X058_pos4_DNA_S42" and "hs_HAMP_A107 *_3'X058_pos2,4_DNA_3'X058_S42" are preferred. [00421] n.d., not determined. [00422] However, further modifications, and/or conjugation to other components, and/or improvement of a delivery vehicle can further improve these molecules.

[00423] Screening of mouse Hepcidin RNAi Agents [00424] 2 1 mm (mouse or M. musculus) Hepcidin RNAi agents are screened, in both 19-mer and 18-mer formats. [00425] Of these, 6 which are active in 18-mer and 19-mer format are picked for additional chemistry and format screens: 254, 256, 257, 280, 281, and 374. [00426] Top mouse candidates (254 and 257) tolerate different stem chemistries and also retain activity with various lipophilic overhangs.

[00427] Some modified variants of these sequences are shown here:

[00428] TABLE 7A. Modified variants of mouse Hepcidin RNAi agents 254, 256, 257, 280, 281, and 374. 56, 65, 76, and 87 indicate: 8 = 2' methoxy ethyl (2 -MOE) T; 7 = 2' methoxy ethyl G; 5 = 2' methoxy ethyl A ; 6= 2' methoxy ethyl C JHU1 115 is a ribitol and phosphate. C6 and C10 are C6 and C10 3' end caps. [00429] Modified variants of Hepcidin RNAi agents include conjugate formats for 18-mers and 19-mers. [00430] Modified variants of Hepcidin RNAi agents include those with modifications of 5' and/or 3' end of the passenger strand to components which increase targeting to and uptake by the liver; and/or modification of the 5' end of the anti-sense strand to stabilize it; and/or modification of the 3' end of the anti-sense strand, e.g., by conjugation with a 3' end cap, to increase stability and/or potency.

[00431] MODIFICATIONS OF STEM CHEMISTRY [00432] As a non-limiting example: Various conjugate formats of 18-mer or 19-mer Hepcidin RNAi agents can comprise, at the 5' end of the sense (passenger) strand, a cholesterol or alpha-tocopherol, GalNac, trivalent GalNac, or HS (hemisuccinate), and/or at the 3' end, in 5' to 3' order, GalNac, C7 (also designated C7-NH2), and a peptide, liver delivering peptide, or transferrin minic (e.g., transferrin, KDEL, TAT, HSP70-derived polypeptide, Arg9, or Melittin). The anti-sense or guide strand can comprise, at the 5' end, 5' stabilizing chemistry, and at the 3' end, a 3' end cap such as a PAZ ligand or other cap, e.g., C6, C10 or biphenyl.

TABLE 7B. [00433] DELIVERY [00434] Any pharmaceutical carrier known in the art can be used to deliver Hepcidin siRNAs.

[00435] Criteria for an effective delivery vehicle include effective target knockdown in the liver at a reasonable dosage and duration, and acceptable pharmacokinetics and toxicity. [00436] Delivery vehicles include, for example, lipid nanoparticles (LNPs), non-LNP delivery, peptide conjugation, and lipoprotein particles (LLPs). Particles can be, for example 10-100 nM in size, and can, for example, self-assemble in aqueous media. [00437] Lipid nanoparticle (LNP) [00438] For example, Hepcidin siRNAs can be delivered using a lipid nanoparticle (LNP). Such an LNP can comprise, for example, a cationic lipid, cholesterol, a neutral lipid, and a shielding lipid. Additional compositions that can be used for delivery of the various RNAi agents are known in the art, e.g., are provided in U.S. Applications No. 61/774759; 61/918,175, filed 12/19/13; 61/918,927; 61/918,182; 61/918941 ; 62/025224; 62/046487; and International

Applications No. PCT/US04/04291 1; PCT/EP201 0/07041 2; PCT/IB201 4/059503. [00439] Several in vivo studies were done using Hepcidin 18-mer RNAi agents at 1 mg/kg administered to mice using LNPs. In one study, Hepcidin RNAi agents were able to deduce gene expression by over 70% compared to PBS control at 48 hours. Other studies demonstrated the efficacy of Hepcidin siRNAs in vivo using a variety of LNPs. [00440] The use of one LNP and an 18-mer Hepcidin RNAi yielded a 60% KD in mice 48 and 96 hrs post IV injection.

[00441] Additional Definitions [00442] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail. [00443] As used throughout this disclosure, articles such as "a" and "an" refer to one or more than one (at least one) of the grammatical object of the article. RNAi agent [00444] In one aspect, the present disclosure pertains to a Hepcidin RNAi agent or other composition comprising at least an antisense nucleic acid sequence complementary to a Hepcidin nucleic acid (or portion thereof), or pertains to a recombinant expression vector encoding an shRNA or composition comprising the antisense nucleic acid that can function as an RNAi as defined below. As used herein, an "antisense" nucleic acid comprises a nucleotide sequence complementary to a "sense" nucleic acid encoding the Hepcidin protein (e.g., complementary to the coding strand of a double-stranded DNA, complementary to an mRNA or complementary to the coding strand of a Hepcidin gene or nucleic acid). [00445] RNAi agents include, as non-limiting examples, siRNAs (small interfering RNAs), dsRNAs (double stranded RNAs), shRNAs (short hairpin RNAs) and miRNAs (micro RNAs). RNAi agents also include, as additional non-limiting examples, locked nucleic acid (LNA), Morpholino, threose nucleic acid (TNA), or glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), peptide nucleic acid (PNA), or UNA (unlocked nucleic acid). RNAi agents also include molecules in which one or more strands are a mixture of RNA, DNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, UNA, and/or FANA, etc. As a non- limiting example, one or both strands of an RNAi agent could be, for example, RNA, except that one or more RNA nucleotides is replaced by DNA, LNA, morpholino, TNA, GNA, UNA, and/or FANA, etc. In various aspects, one or both strands of the RNAi agent can be nicked, and both strands can be the same length, or one (e.g., the passenger strand), can be shorter than the other. [00446] In various aspects, the present disclosure pertains to any RNAi agent comprising a RNA sequence disclosed herein and/or a RNA sequence corresponding to any DNA sequence disclosed herein (e.g., wherein the DNA nucleotides are replaced by the corresponding RNA nucleotide, for example, with T in DNA replaced by U in RNA, and with ribose instead of deoxyribose in the sugar-phosphate backbone). [00447] The RNAi agent(s) of the present disclosure target (e.g., bind to, anneal to, hybridize, etc.) the Hepcidin mRNA. The use of the RNAi agent specific to Hepcidin results in a decrease of Hepcidin activity, level and/or expression, e.g., a "knock-down" (KD) or "knock-out" of the target gene or target sequence. In one aspect, in the case of a disease state characterized by over-expression, over-abundance or hyper-activity of Hepcidin, administration of a RNAi agent to Hepcidin knocks down the Hepcidin target enough to provide a more normal or therapeutic level of Hepcidin activity or expression. Hepcidin -/- mice showed multiple organ pathology, biochemical abnormalities and altered gene expression; see Scortegagna et al. Nat. Genet. 35: 331-340. Thus, a minimal expression of Hepcidin in normal tissues can be beneficial. In various aspects of the disclosure, the patient or individual may have a disease state characterized by excessively high levels of Hepcidin and the RNAi agent can restore a normal level. In one aspect of the disclosure, the levels of Hepcidin throughout the body are modulated such that Hepcidin levels in one area (e.g., areas afflicted by a Hepcidin-related disease) are lower, while areas of the body not afflicted by the disease are closer to normal

Hepcidin levels. In one aspect of the disclosure, the RNAi agent can be delivered locally so that levels of Hepcidin outside the diseased areas can be maintained as close to normal as possible. In another aspect, the level of Hepcidin in the body can be modulated such that it is low enough to improve the disease state, but not so low that organ pathology occurs.

[00448] In one aspect, the RNAi comprises a single strand. This single-stranded RNAi agent oligonucleotide or polynucleotide can comprise the sense or antisense strand, as described by Sioud 2005 J. Mol. Biol. 348:1079-1090, and references therein. Thus the disclosure encompasses RNAi agents with a single strand comprising either the sense or antisense strand of an RNAi agent described herein. The disclosure also encompasses RNAi agents comprising a single strand, wherein the single strand comprises the sequences of both the antisense and sense strands of any RNAi agent disclosed herein, e.g., wherein the strands are contiguous, connected by a loop or otherwise linked. Examples of such molecules include those with a hairpin between the sense and anti-sense sequences (e.g., shRNA).

[00449] In various aspects, one or both strands contain one or more nicks, i.e., a break or missing bond in the phosphate backbone, such that at least one nucleotide subunit is not covalently linked to the adjacent nucleotide subunit in any given sequence. In some aspects, the passenger strand is nicked (see, for example, WO 2007/107162). In various aspects, one or both strands contain one or more gaps, e.g., wherein at least one entire nucleotide subunit is absent from the disclosed sequence. Where a sense or antisense sequence contains a gap, that strand is envisioned to comprise two separate oligonucleotides. [00450] Particularly useful siRNAs include those which can bind specifically to those regions of the Hepcidin mRNA that have one or more of the following qualities: binding in the coding segment of Hepcidin; binding at or near the junction of the 5' untranslated region and the start of the coding segment; binding at or near the translational start site of the mRNA; binding at, across or near junctions of exons and introns; little or no binding to the mRNAs or transcripts of other genes (little or no "off-target effects"); binding to the Hepcidin mRNA in or near a region or regions that is not double-stranded or a stem region, e.g., those in a loop or single-stranded portion; eliciting little or no immunogenicity; binding in a segment of the Hepcidin mRNA sequence which is conserved among various animal species (including human, mouse, rat, cyno, etc.), as the presence of a conserved sequence facilitates testing using various laboratory animals; binding to double-stranded region(s) of the mRNA; binding to an AT-rich region (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60% AT-rich); and/or lacking particular sequences known or suspected to decrease siRNA activity, e.g., the presence of a GG sequence at the 5' end, which may decrease separation of the double-stranded portion of the siRNA. In one aspect, the RNAi agent specific to Hepcidin can be a double-stranded RNA having any one or more of these qualities. [00451] The term "double-stranded RNA" or "dsRNA," as used herein, refers to a RNAi agent comprising a first and a second strand; e.g., a composition that includes an RNA molecule or complex of molecules having a hybridized duplex region (i.e., a region where the nucleotide bases from the first strand and the second strand are paired) that comprises two anti- parallel and substantially complementary nucleic acid strands, which will be referred to as having "sense" and "antisense" orientations with respect to a target RNA. The antisense strand, with respect to the mRNA target, is also called the "guide" strand, and the sense strand is also called the "passenger" or "anti-guide" strand. The passenger strand can include at least one or more of the following: one or more extra nucleotides (e.g., a bulge or 1 nt loop) compared to the other strand, and/or a nick, a gap, a mismatch, etc., compared to the other strand. In various aspects, the RNAi agent comprises a first strand and a second strand. In various aspects, and as used herein and as is clear by context, terminology referring to the first strand refers to the sense strand and the second strand refers to the anti-sense strand as listed in any Table herein.

In other aspects, and as used herein and as is clear by context, the first strand refers to the anti- sense strand, and the second strand refers to the sense strand as listed in any Table herein. [00452] The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs ("bp") in length, e.g., 15-30 bp in length. Considering a duplex between 9 and 36 bp, the duplex can be any length in this range, for example, 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 bp and any sub-range therebetween, including, but not limited to 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30 bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 20 basepairs, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 21-23 bp, 21-22 bp, 2 1 bp, 22 bp, or 23 bp. The dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of about 19 to about 22 bp in length, though the strands of artificial dsRNAs can be shorter or longer. siRNAs wherein one or both strands are as short as 16 or 15 nt still demonstrate RNA interference activity. Chu and Rana 2008 RNA 14: 1714-1719. One strand of the duplex region of a dsRNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary duplex region, or can be formed from two or more separate RNA molecules that hybridize to form the duplex. Where the duplex region is formed from two self-complementary regions of a single molecule, the molecule can have a duplex region separated by a single-stranded chain of nucleotides (herein referred to as a "hairpin loop", e.g., such as found in an shRNA construct) between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some aspects 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. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can, be covalently connected. Where the two strands are connected covalently by a hairpin loop, the construct is generally referred to herein and in the art as a "shRNA". Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker."

[00453] RNAi Agents to Hepcidin comprising mismatches from the disclosed sequences [00454] Various specific aspects of a RNAi agent to Hepcidin are disclosed herein; example sequences are provided in the Tables. Specific aspects of the present disclosure include RNAi agents which comprise sequences differing by 0, 1, 2 , or 3 nt (nucleotides) or bp [basepair(s)]

(e.g., with 0, 1, 2 or 3 mismatches) from any of the RNAi agents listed in any of Tables 1 to 4 and 6, and modified and unmodified variants thereof. [00455] A mismatch is defined herein as a difference between the base sequence or length when two sequences are maximally aligned and compared. A mismatch is defined as a position wherein the base of one sequence does not match the base of the other sequence. Thus, a mismatch is counted, for example, if a position in one sequence has a particular base (e.g., A), and the corresponding position on the other sequence has a different base (e.g., G). Substitution of A , for example, with T, C, G or U would constitute a mismatch. Substitution of G with T, A , C or U would also constitute a mismatch. Substitution of C with T, G, A or U would also constitute a mismatch. Substitution of U with A , C or G would constitute a mismatch. Note, however, that on a given strand, a U can be replaced by T (either as RNA or, preferably, DNA, e.g., 2'-deoxy-thymidine); the replacement of a U with a T is not a mismatch as used herein, as either U or T can pair with A on the opposite strand. The RNAi agent can thus comprise one or more DNA bases, e.g., T. In some cases, in a portion or portions of the RNAi agent, DNA can be used in place of RNA (e.g., in the seed region), to form a DNA-RNA hybrid. See, for example, Yamato et al. 201 1 cancer Gene Ther. 18: 587-597. No mismatch is counted between a DNA portion(s) of the RNAi agent and the corresponding target mRNA if basepairing occurs (e.g., between A , G, C, or T in the DNA portion, and the corresponding U, C, G, or A , respectively in the mRNA). [00456] A mismatch is also counted, e.g., if a position in one sequence has a base (e.g., A), and the corresponding position on the other sequence has no base (e.g., that position is an abasic nucleotide, which comprises a phosphate-sugar backbone but no base). A single- stranded nick in either sequence (or in the sense or anti-sense strand) is not counted as mismatch. Thus, as a non-limiting example, no mismatch would be counted if one sequence comprises the sequence AG, but the other sequence comprises the sequence AG with a single- stranded nick between the A and the G . A nucleotide modification in the sugar or phosphate is also not considered a mismatch. Thus, if one sequence comprises a C, and the other sequence comprises a modified C (e.g., 2'-modification) at the same position, no mismatch would be counted. [00457] Thus, no mismatches are counted if modifications are made to the sugar, phosphate, or backbone of the RNAi agent without modifying the base. Thus, a strand having the sequence of UAUUCCAAGACCUAUGUU (SEQ ID NO: 87) as an RNA would have zero mismatches from another strand having the same sequence as a PNA; or morpholino; or LNA; or TNA; or GNA; or UNA; or FANA; or a mix or chimera of RNA and DNA, TNA, GNA, UNA, FANA, morpholino, LNA, and/or PNA, etc.

[00458] It is also noted that the sequences of the RNAi agents in the Tables include sequences which comprise modifications. It is noted that dTdT (2'-deoxy-thymidine-5'- phosphate and 2'-deoxy-thymidine-5'-phosphate), or in some cases, TT or UU, can be added as a terminal dinucleotide cap or extension to one or both 3'-ends, but this cap or extension is not included in the calculation of the total number of mismatches and is not considered part of the target sequence. This is because the terminal dinucleotide protects the ends from nuclease degradation but does not contribute to target specificity (Elbashir et al. 2001 Nature 4 11: 494- 498; Elbashir et al. 2001 EMBO J. 20: 6877-6888; and Kraynack et al. 2006 RNA 12:163-176).

[00459] In addition, a modified variant can have one or more modifications from the corresponding unmodified sequence. In this case, lowercase "c" represents 2'-0- methylcytidine-5'-phosphate, and lowercase "u" represents 2'-0-methyluridine-5'-phosphate. Uppercase "A", "C", "G" and "U" represent the un-modified adenosine-5'-phosphate, cytidine-5'- phosphate, guanosine-5'-phosphate, and uridine-5'-phosphate, respectively. Various modifications are shown in Figure 1. The substitution, for example, of modified c for unmodified

C does not count as a mismatch in numbering the 0, 1, 2, or 3 mismatches between sequences. This nomenclature is used for all sequences in any of Tables 1 to 4 and 6. Thus, an equal number of mismatches would be calculated (a) between a test sequence and that of another RNAi agent, and (b) between the same test sequence and the corresponding unmodified sequence from the Hepcidin gene, and (c) between a modified sequence and a differently modified sequence which have the same base sequence. [00460] In one particular aspect, the present disclosure comprises a RNAi agent comprising a anti-sense strand comprising at least 15 to 19 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of the anti-sense strand of: any of the RNAi agents listed in any of Tables

1 to 4 and 6, and modified and unmodified variants thereof. [00461] The present disclosure pertains to "modified and unmodified variants" of the disclosed sequences. [00462] An "unmodified variant" of a particular sequence is the corresponding portion of Hepcidin without any modifications. Example modified sequences are disclosed herein The "unmodified variants" of the sequences of the Tables have the identical sequence, without base modifications or terminal dTdT. A given sequence and an "unmodified variant" of it differ by 0 nt (and have no mismatches). [00463] A "modified variant" of a particular sequence comprises one or more (or one or more fewer) modifications to the backbone, sugar, phosphate or base, and/or addition of a terminal dinucleotide (e.g., TT, dTdT, TsT or UU), but do not have any base substitutions (e.g., G for C, or A for G); thus a given sequence and a modified variant thereof differ by 0 nt (and have no mismatches). As another example, a given sequence as a RNA and the same sequence as a PNA are modified variants of each other and differ by 0 nt (and have no mismatches). Similarly, the same sequence (with no base substitutions) as a locked nucleic acid (LNA), Morpholino, threose nucleic acid (TNA), or glycol nucleic acid (GNA) or unlocked nucleic acid (UNA) or FANA or ANA or HNA or CeNA, etc. would be a modified variant which has 0 mismatches. In addition, the same sequence could be used in strands which are a mixture of RNA, DNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, UNA, and/or FANA, etc. As a non-limiting example, one or both strands could be, for example, RNA except that one or more nucleotides is replaced by DNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, UNA, and/or FANA, etc. [00464] As detailed below, substituting a single nucleotide at a given position with a modified version of the same nucleotide would produce a modified variant (with 0 mismatches). [00465] In another particular aspect, the RNAi agent comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand of any of the RNAi agents listed in the Tables and modified and unmodified variants thereof. [00466] RNAi agents to Hepcidin of the present disclosure can be used in RNA interference.

[00467] Modifications of RNAi Agents [00468] The present disclosure encompasses both unmodified and example modified RNAi agents, such as those disclosed in the Tables. [00469] The present disclosure further encompasses any other modification of a disclosed RNAi agent (e.g., a modified variant). [00470] For example, the disclosure encompasses a RNAi agent with a substitution of a single nucleotide at a given position with a modified version of the same nucleotide. Thus a nucleotide (A, G, C or U) can be replaced by the corresponding 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2- thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, or 2,6-diaminopurine. [00471] Additional modified variants include the addition of any other moiety (e.g., a radiolabel or other tag or conjugate) to the RNAi agent; provided that the base sequence is identical, the addition of other moieties produces a "modified variant" (with no mismatches). [00472] Various sets of modifications can be used. These include the following formats, which are used in various screens disclosed herein.

Two such sets of modifications include A51 S26 and A85S26.

A51S53

Guide strand: All U and C except positions 1, 2 and 14 are 2'-OMe Sense strand: All U as 2'-OMe-U.

A85S26 ("NEW")

Guide strand: All U except position 1, 2 and 14 are 2'-OMe Sense strand: All C and U are modified (2'-OMe)

However, other modifications of the Hepcidin RNAi agent sequences disclosed herein could be prepared. These include, as non-limiting examples:

A51 S26

A51 : In guide strand all U as 2'-OMe-U and all C as 2'-OMe-C, except pos. 1, 2 and 14 ; 3' overhangs as 2'-OMe-U 2'-OMe-U

S26: In sense strand all U as 2'-OMe-U and all C as 2'-OMe-C; 3' overhangs as 2'-OMe-U 2'- OMe-U

A22S26 A22 indicates that All UA as 2'-OMe-U A and all CA as 2'-OMe-C A S26 indicates that All U as 2'-OMe-U and all C as 2'-OMe-C All 3' overhangs as 2'-OMe-U 2'-OMe-U

[00473] In addition to these modifications and patterns (e.g., formats) for modifications, other modifications or sets of modifications of the sequences provided can be generated using common knowledge of nucleic acid modification. These various aspects of the RNAi agents to Hepcidin of the present disclosure can be used in RNA interference.

[00474] RNA Interference [00475] RNA interference (RNAi) is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA. The process of RNAi occurs when ribonuclease III (Dicer) cleaves the longer dsRNA into shorter fragments called siRNAs. siRNAs (small interfering

RNAs) produced by Dicer are typically about 2 1 to 23 nucleotides long and comprise about 19 base pair duplexes (though artificial siRNAs or RNAi agents can be shorter or longer, and/or blunt-ended, and/or comprises one or more endcaps). The smaller RNA segments then mediate the degradation of the target mRNA. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control. Hutvagner et al. 2001 Science 293: 834. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded mRNA complementary to the anti-sense strand of the RNAi agent. Cleavage of the target RNA takes place in the middle of the region complementary to the anti-sense strand of the siRNA duplex.

[00476] In one aspect, an RNA interference agent includes a single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, the present disclosure contemplates a long double-stranded RNA introduced into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer. Sharp et al. 2001 Genes Dev. 15:485. Dicer, a ribonuclease-lll- like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs. Bernstein, et al. 2001 Nature 409:363. The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling one of the now unpaired siRNA strands to act as a "guide" strand to guide target recognition. Nykanen, et al. 2001 Cell 107:309. Upon binding of the antisense guide strand to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing. Elbashir, et al. 2001 Genes Dev. 15:188. Thus, in one aspect the present disclosure relates to a single-stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene. [00477] Kits for RNAi synthesis are commercially available, e.g., from New England Biolabs and Ambion.

[00478] A suitable RNAi agent can be selected by any process known in the art or conceivable by one of ordinary skill in the art. For example, the selection criteria can include one or more of the following steps: initial analysis of the Hepcidin gene sequence and design of RNAi agents; this design can take into consideration sequence similarity across species (human, cynomolgus, mouse, etc.) and dissimilarity to other (non-Hepcidin) genes; screening of

RNAi agents in vitro (e.g., at 10 nM in RKO cells); determination of EC50 in RKO cells; determination of viability of cells treated with RNAi agents, including insensitive cells which do not require Hepcidin for survival, or sensitive cells, which do require Hepcidin for survival; testing with human PBMC (peripheral blood mononuclear cells), e.g., to test levels of TNF-alpha to estimate immunogenicity, wherein immunostimulatory sequences are less desired; testing in human whole blood assay, wherein fresh human blood is treated with an RNAi agent and cytokine/chemokine levels are determined [e.g., TNF-alpha (tumor necrosis factor-alpha) and/or MCP1 (monocyte chemotactic protein 1)], wherein Immunostimulatory sequences are less desired; determination of gene knockdown in vivo using subcutaneous tumors in test animals; Hepcidin target gene modulation analysis, e.g., using a pharmacodynamic (PD) marker, for example, other factors whose expression is affected by Hepcidin, wherein Hepcidin knockdown leads to a dose-dependent reduction of abundance of those components; and optimization of specific modifications of the RNAi agents. [00479] The dsRNA molecules (RNAi agents) described herein are thus useful in RNA interference of Hepcidin.

[00480] Features of a RNAi Agent: Sense strand, Antisense Strand and (Optional) Overhangs [00481] In various aspects, the RNAi agents comprise a first strand and a second strand, e.g., a sense strand and an antisense strand (or an antisense and a sense strand), optionally, either or both both ends of either or both strand can comprise unpaired nucleotides (referred to herein as "overhangs"). [00482] The term "antisense strand" refers to the strand of a RNAi agent which includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. [00483] The term "sense strand," as used herein, refers to the strand of a RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein. [00484] The sequence of a gene may vary from individual to individual, especially at wobble positions within the coding segment, or in the untranslated region; individuals may also differ from each other in coding sequence, resulting in additional differences in mRNA. The sequence of the sense and antisense strands of the RNAi agent can thus be designed to correspond to that of an individual patient, if and where needed. RNAi agents can also be modified in sequence to reduce immunogenicity, binding to undesired mRNAs (e.g., "off-target effects") or to increase stability in the blood. These sequence variants are independent of chemical modification of the bases or 5' or 3' or other end-caps of the RNAi agents.

[00485] The RNAi agents can also have overhangs of 0, 1, or 2 overhangs; in the case of a 0 nt overhang, they are blunt-ended. A RNAi agent can thus have 0, 1 or 2 blunt ends. In a

"blunt-ended RNAi agent" both strands terminate in a base-pair; thus a blunt-ended molecule lacks either 3' or 5' single-stranded nucleotide overhangs. [00486] The RNAi agents can comprise overhang(s), blunt end(s), and/or 5' and 3' endcap(s). [00487] As used herein, the term "overhang" or "nucleotide overhang" refer to at least one unpaired nucleotide that protrudes from the end of at least one of the two strands of the duplex structure of a RNAi agent. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, this forms a nucleotidic overhang, e.g., the unpaired nucleotide(s) form the overhang. 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, at least five nucleotides or more. An overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5' end, 3' end or both ends of either an antisense or sense strand of a dsRNA.The RNAi agent can also optionally comprise a cap. The term "Cap" and the like include a chemical moiety attached to the end of a double- stranded nucleotide duplex, but is used herein to exclude a chemical moiety that is a nucleotide or nucleoside. A "3' Cap" is attached at the 3' end of a nucleotide or oligonucleotide and protects the molecule from degradation, e.g., from nucleases, such as those in blood serum or intestinal fluid. A non-nucleotidic 3' cap is not a nucleotide and can replace a TT or UU dinucleotide at the end of a blunt-ended RNAi agent. In one aspect, non-nucleotidic 3' end caps are as disclosed in, for example, WO 2005/021749 and WO 2007/128477; and U.S. Pat. No. 8,097,716; U.S. Pat. No. 8,084,600; and U.S. Pat. No. 8,344,128. A "5' cap" is attached at the 5' end of a nucleotide or oligonucleotide. A cap should not interfere (or unduly interfere) with RNAi activity.

[00488] The present disclosure thus contemplates a RNAi agent specific to Hepcidin comprising an antisense strand (which may be contiguous or connected via a linker or loop) in a

RNAi agent. In a more specific aspect, an RNAi agent comprises an antisense strand and a sense strand which together comprise a double-stranded or complementary region. In one aspect, it can also optionally comprise one or two overhangs and/or one or two caps. The RNAi agent is used to induce RNA interference of the target gene, Hepcidin.

[00489] Target and complementary sequences [00490] The RNAi agents of the present disclosure target (e.g., specifically bind to, anneal to, etc.) the mRNA encoding Hepcidin. The use of the RNAi agent specific to Hepcidin results in a decrease of Hepcidin activity, level and/or expression. Particularly in one aspect, in the case of a disease state characterized by over-expression, over-abundance or hyper-activity of Hepcidin, administration of a RNAi agent to Hepcidin knocks down the Hepcidin gene enough to restore a normal level of Hepcidin activity or expression.

[00491] In one aspect, the first or second strand of the RNAi comprises a sequence complementary to that of the target nucleic acid, Hepcidin. [00492] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. [00493] As used herein, "target sequence" or "target gene" refer to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, e.g., a Hepcidin gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides ("nt") in length, e.g., 15-30 nt in length, including all sub ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nt, 15- 26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18- 23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 19-30 nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19-20 nt, 19 nt, 20-30 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21 nt, 20 nt, 21-30 nt, 21-26 nt,

21-25 nt, 21-24 nt, 21-23 nt, or 21-22 nt, 2 1 nt, 22 nt, or 23 nt. The sense and antisense strands of the RNAi comprise a sequence complementary to that of the target nucleic acid, Hepcidin. [00494] As used herein, and unless otherwise indicated, the term "complementary" refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such conditions can, for example, be stringent, e.g., 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. [00495] Complementary sequences within a RNAi agent, e.g., within a dsRNA as described herein, include base-paired oligonucleotides or polynucleotides comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4 , or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single-stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide

2 1 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 2 1 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes described herein. The term "overhang" describes an unpaired nucleotide at the 3' or 5' end of a double-stranded nucleotide duplex, as described above. In one aspect, the overhang is 1 to 4 nt long and is on the 3' end. [00496] "Complementary" sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, Wobble or Hoogstein base pairing. The terms "complementary," "fully complementary" and "substantially complementary" herein may furthermore be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a RNAi agent and a target sequence, as will be understood from the context of their use. As used herein, a polynucleotide that is "substantially complementary to at least part of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding Hepcidin). For example, a polynucleotide is complementary to at least a part of a Hepcidin mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Hepcidin. [00497] Thus, the RNAi agent of the present disclosure is complimentary or substantially complimentary to a target sequence in the target Hepcidin and is double-stranded, comprising a sense and an antisense strand (which can be contiguous, linked via a loop, or otherwise joined), where the double-stranded region an be 9 to 36 bp long (particularly for example, 19-22 bp or 19-23 bp long), and can furthermore optionally comprise a 3' or 5' overhang, and the RNAi agent can furthermore comprise a 3' cap. The RNAi agent mediates RNA interference, down- regulating or inhibiting or reducing the level, expression and/or activity of Hepcidin, and/or establishing or re-establishing an approximately normal level of Hepcidin and/or Hepcidin activity, or other biological function related to Hepcidin. [00498] Thus, the RNAi agent of the present disclosure is complimentary or substantially complimentary to a target sequence in the target Hepcidin and is double-stranded, comprising a sense and an antisense strand (which can be contiguous, linked via a loop, or otherwise joined), where the double-stranded region an be 9 to 36 bp long (particularly for example, 19-22 bp or 19-23 bp long), and can furthermore optionally comprise a 3' or 5' overhang, and the RNAi agent can furthermore comprise a 3' cap. The RNAi agent mediates RNA interference, down- regulating or inhibiting the level, expression and/or activity of Hepcidin, and/or establishing or re establishing an approximately normal level of Hepcidin activity or expression. [00499] The term "double-stranded RNA" or "dsRNA," as used herein, refers to an RNAi agent comprising a first and a second strand; e.g., a composition that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti- parallel and substantially complementary nucleic acid strands, which will be referred to as having "sense" and "antisense" orientations with respect to a target RNA. The antisense strand, with respect to the mRNA target, is also called the "guide" strand, and the sense strand is also called the "passenger" strand. As used herein, depending on the context, the "first" strand can be the guide or antisense strand, and the "second" strand can be the passenger or sense strand. Also as used herein, again depending on the context, the "first" strand can be the passenger or sense strand, and the "second" strand can be the guide or antisense. The passenger strand can include at least one or more of the following: one or more extra nucleotides (e.g., a bulge or 1 nt loop) compared to the other strand, a nick, a gap, etc., compared to the other strand. In various aspects, the first strand is the sense strand and the second strand is the anti-sense strand. In other aspects, the first strand is the anti-sense strand, and the second strand is the sense strand. [00500] The duplex region can be of any length that permits loading into the RISC complex and subsequent specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs ("bp") in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

3 1, 32, 33, 34, 35, or 36 bp and any sub-range therebetween, including, but not limited to 15-30 base pairs, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp, 19- 22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30 bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp,

20-21 bp, 20 bp, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 21-23 bp, 21-22 bp, 2 1 bp, 22 bp, or 23 bp. [00501] The dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of about 19 to about 22 base pairs in length, although artificial RNAi agents can be synethesized or made by any method known in the art. One strand of the duplex region of a dsRNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary duplex region, or can be formed from two or more separate RNA molecules that hybridize to form the duplex. Where the duplex region is formed from two self-complementary regions of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a "hairpin loop", e.g., such as found in an shRNA construct) between the 3'-end of one strand and the 5'- end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some aspects 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. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by a hairpin loop, the construct is generally referred to herein and in the art as a "shRNA". Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" is also used herein to refer to a dsRNA as described above.

[00502] RNAi Agents Lowering or Normalizing Hepcidin Level, Expression And/Or Activity [00503] RNAi agents for targeting Hepcidin include those which bind to a Hepcidin sequence provided herein and which work to reduce Hepcidin through a RNAi mechanism. Example RNAi agents (e.g., siRNAs) to Hepcidin are provided, e.g., in any of Tables 1 to 4 and 6 . [00504] Any method known in the art can be use to measure changes in Hepcidin activity, level, and/or expression induced by a Hepcidin RNAi agent. Measurements can be performed at multiple timepoints, prior to, during and after administration of the RNAi agent, to determine the effect of the RNAi agent. [00505] The RNAi agents of the present disclosure silence, inhibit the expression of, down- regulate the expression of, and/or suppress the expression of Hepcidin, such that an approximately normal level of Hepcidin activity or expression is restored.

[00506] In addition, in various aspects, depending on the disease condition and biological context, it is acceptable to use the RNAi agents of the present disclosure to establish a level of Hepcidin expression, activity and/or level which is below the normal level, or above the normal level, depending on the therapeutic outcome that is desired. [00507] Any method known in the art can be use to measure changes in Hepcidin activity, level and/or expression induced by a Hepcidin siRNA. Measurements can be performed at multiple timepoints, prior to, during and after administration of the siRNA, to determine the effect of the siRNA. [00508] The terms "silence," "inhibit the expression of," "down-regulate the expression of," "suppress the expression of," and the like, in so far as they refer to a Hepcidin gene, herein refer to the at least partial suppression of the expression of a Hepcidin gene, as manifested by a reduction of the amount of Hepcidin mRNA which may be isolated from or detected in a first cell or group of cells in which a Hepcidin gene is transcribed and which has or have been treated such that the expression of a Hepcidin gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

[00509] Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Hepcidin gene expression, e.g., the amount of protein encoded by a Hepcidin gene, alteration in expression of a protein whose expression is dependent on Hepcidin, etc. In principle, Hepcidin gene silencing may be determined in any cell expressing Hepcidin, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference or control is needed in order to determine whether a given RNAi agent inhibits the expression of Hepcidin by a certain degree and therefore is encompassed by the instant disclosure, the assays provided in the Examples below shall serve as such reference. [00510] For example, in certain instances, expression of Hepcidin is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a RNAi agent featured in the present disclosure. In some aspects, Hepcidin is suppressed by at least about

60%, 70%, or 80% by administration of a RNAi agent featured in the present disclosure. In some aspects, Hepcidinjs suppressed by at least about 85%, 90%, or 95% or more by administration of a RNAi agent, as described herein. In one aspect, the degree of Hepcidin suppression is determined by loss of full length Hepcidin mRNA in a treated cell compared to an untreated cell. In one aspect, the degree of Hepcidin suppression is determined with a phenotypic assay that monitors loss of proliferative activity and/or cell death. Other aspects are as provided in the Examples. [00511] A suitable RNAi agent can be selected by any process known in the art or conceivable by one of ordinary skill in the art. For example, the selection criteria can include one or more of the following steps: initial analysis of the Hepcidin gene sequence and design of RNAi agents; this design can take into consideration sequence similarity across species (human, cynomolgus, mouse, etc.) and dissimilarity to other (non-Hepcidin) genes; screening of RNAi agents in vitro (e.g., at 10 nM and 1 nM in Huh-7 cells); selection of RNAi agents with high knock-down at 10 nM and 1 nM in Huh-7 cells; determination of EC50 in Huh-7 cells; confirmation of EC50 in a RCC cell line (Huh-7 cells); analysis of a lack of effect on cell growth relative to a control siRNA; reduction in Huh-7 cells of expression of a HRE-luc (luciferase) reporter gene and not control UB6-luc (luciferase) reporter gene; Western blots to measure Hif-

1, Hif-2, and ARNT levels; testing with human PBMC (peripheral blood mononuclear cells), e.g., to test levels of TNF-alpha to estimate immunogenicity, wherein immunostimulatory sequences are less desired; testing in human whole blood assay, wherein fresh human blood is treated with an RNAi agent and cytokine/chemokine levels are determined [e.g., TNF-alpha (tumor necrosis factor-alpha) and/or MCP1 (monocyte chemotactic protein 1)], wherein immunostimulatory sequences are less desired; determination of gene knockdown in vivo using subcutaneous tumors in test animals; Hepcidin target gene modulation analysis, e.g., using a pharmacodynamic (PD) marker, for example, EGLN3, SLC2A1 and VEGF, wherein Hepcidin knockdown leads to a dose-dependent alteration of EGLN3, SLC2A1 and VEGF expression in cells; and optimization of specific modifications of the RNAi agents. As appropriate, other cell lines can be used in place of those listed above to identify RNAi agents capable of lowering Hepcidin levels or decrease symptoms of a Hepcidin-related disease. [00512] By "lower" in the context of Hepcidin or a symptom of a Hepcidin-related disease is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more. If, for a particular disease, or for an individual suffering from a particular disease, the levels or expression of Hepcidin are elevated, treatment with a Hepcidin RNAi agent of the present disclosure can particularly reduce the level or expression of Hepcidin to a level considered in the literature as within the range of normal for an individual without such disorder, or to a level that reduces or ameliorates symptoms of a disease. The level or expression of Hepcidin can be measured by evaluation of mRNA (e.g., via Northern blots or PCR), or protein (e.g., Western blots). The effect of a RNAi agent on Hepcidin expression can be determined by measuring Hepcidin gene transcription rates (e.g., via Northern blots; or reverse transcriptase polymerase chain reaction or real-time polymerase chain reaction).

[00513] As used herein, "down-regulates" refers to any statistically significant decrease in a biological activity and/or expression of Hepcidin, including full blocking of the activity (i.e., complete inhibition) and/or expression. For example, "down-regulation" can refer to a decrease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % in Hepcidin level, activity and/or expression. [00514] As used herein, the term "inhibit" or "inhibiting" Hepcidin refers to any statistically significant decrease in biological level, activity and/or expression of Hepcidin, including full blocking of the activity and/or expression. For example, "inhibition" can refer to a decrease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % in Hepcidin level, activity and/or expression. As used herein, the term "inhibit" similarly refers to a significant decrease in level, activity and/or expression, while referring to any other biological agent or composition. [00515] By "level", it is meant that the Hepcidin RNAi agent can alter the level of Hepcidin, e.g., the level of Hepcidin mRNA or the level of Hepcidin protein, or the level of activity of Hepcidin.

[00516] Some diseases include any Hepcidin-related disease disclosed herein or known in the literature. Particularly in one aspect, in the case of a disease characterized by over- expression and/or hyper-activity of Hepcidin, administration of a RNAi agent to Hepcidin reduces the level, expression and/or activity of Hepcidin. Thus, in various aspects, administration of a RNAi agent to Hepcidin particularly establishes or re-establishes a normal or approximately normal level of Hepcidin activity, expression and/or level. [00517] By "normal" or "approximately normal" in terms of level, expression and/or activity, is meant at least: about 50%, about 60%, about 70%, about 80%, about 90%, and/or about 100%; and/or no more than: about 100%, about 120%, about 130%, about 140%, or about 150% of the level, expression and/or activity of Hepcidin in a healthy cell, tissue, or organ. This can be measured using, for example, lung or kidney homogenates, as described in Gambling et al. 2 Kidney Intl. 65: 1774-1781 . Particularly in one aspect, administration of the appropriate amount of the appropriate Hepcidin RNAi agent restores Hepcidin level, activity and/or expression to about 50% to about 150%, more particularly about 60% to about 140%, more particularly to about 70% to about 130%, more particularly to about 80% to about 120%, more particularly to about 90% to about 110%, and most particularly to about 100% of that of a healthy cell, tissue or organ. Administration of a Hepcidin RNAi to a patient with a Hepcidin-related disease thus particularly restores the level, activity, and/or expression of Hepcidin and the level of Na+ reabsorption to an approximately normal level, as determined by direct measurements of Hepcidin mRNA or protein levels, or indirect determinations. In addition, the preferred target amount of Hepcidin level, expression and/or activity after Hepcidin RNAi agent administration can be calculated to take into account any other perturbations in a Hepcidin-related pathway. For example, if another factor in a Hepcidin-related pathway is either over- or under-expressed, Hepcidin level, expression or activity may be modulated to attain a more normal state.

[00518] In addition, in various aspects, depending on the disease condition and biological context, it is acceptable to use the RNAi agents of the present disclosure to establish a level of Hepcidin expression, activity and/or level which is below the normal level, or above the normal level.

[00519] Types of RNAi Agents and Modification Thereof [00520] The use of RNAi agents or compositions comprising an antisense nucleic acid to down-modulate the expression of a particular protein in a cell is well known in the art. A RNAi agent comprises a sequence complementary to, and is capable of hydrogen bonding to, the coding strand of another nucleic acid (e.g., an mRNA). Thus, in various aspects, the RNAi agents of the present disclosure encompass any RNAi agents which target (e.g., are complementary, capable of hybridizing or hydrogen bonding to, etc.) any sequence presented, e.g., in any of the Tables. [00521] Once a functional guide strand has been identified, many variations to the guide and/or passenger strand can be made. For example, the RNAi agent may have modifications internally, or at one or both ends. The modifications at the ends can help stabilize the RNAi agent, protecting it from degradation by nucleases in the blood. The RNAi agents may optionally be directed to regions of the Hepcidin mRNA known or predicted to be near or at splice sites of the gene. [00522] A RNAi agent can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, RNAi agent can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to decrease off-target effects, and/or increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. [00523] "G," "C," "A," "T" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, the terms "ribonucleotide", "deoxynucleotide", or "nucleotide" can also refer to a modified nucleotide or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the present disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the present disclosure. [00524] The skilled artisan will recognize that the term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules as expressed or found in nature (i.e., are naturally occurring), but also non-naturally occurring analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. The RNA can be modified in the nucleobase structure or in the ribose- phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives can retain the ability to form a duplex. As non- limiting examples, either or both strand of an RNAi agent can comprise at least one modified ribonucleoside, including but not limited to a 2'-0-methyl modified nucleotide, 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, 2'-alkyl-modified nucleoside, morpholino nucleoside, an unlocked ribonucleotide (e.g., an acyclic nucleotide monomer, as described in WO 2008/147824), a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, 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, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one aspect, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway. In addition, the RNAi agent can comprise one or two strands which are a RNA, or a mixture of RNA, DNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, UNA, and/or FANA, etc. As a non-limiting example, one or both strands could be, for example, RNA except that one or more nucleotides is replaced by DNA, LNA, morpholino, TNA, GNA, ANA, HNA, CeNA, FANA, and/or UNA, etc. [00525] Examples of modified nucleotides which can be used to generate the RNAi agent include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6- diaminopurine. These and other example modifications are shown in Figure 1, herein, and

Usman et al. 1992 TIBS 17:34; Usman et al. 1994 Nucl. Acids Symp. Ser. 3 1: 163; Burgin et al. 1996 Biochem. 35: 14090. [00526] A "modified variant" of a sequence disclosed herein includes any variant comprising the same sequence, but with a modification in the base, sugar, phosphate or backbone (but not a base substitution, e.g., A for G, or C for U). Thus, a modified variant can comprise any modified nucleotide described above (e.g., 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, etc.). When a base is replaced by a corresponding modified base (e.g., A for modified A), these modified nucleotides do not constitute a mismatch or base difference. Thus a given sequence with a U at a particular position and a modified variant comprising a 5-fluorouracil, 5- bromouracil, 5-chlorouracil, or 5-iodouracil at the same sequence would differ by 0 nt (or have no mismatches); however, a given sequence with a C at a particular position and a different sequence with a 5-fluorouracil (wherein the two sequences are otherwise identical) would differ by 1 nt ( 1 mismatch). [00527] Replacing the 3'-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3'-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity. International PCT Publication No. WO 00/44914, and Beach et al. International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom. Kreutzer et al. Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2'-amino or 2'-0-methyl nucleotides, and nucleotides containing a 2'-0 or 4'-C methylene bridge. Additional 3'-terminal nucleotide overhangs include dT (deoxythimidine), 2'- 0,4'-C-ethylene thymidine (eT), and 2-hydroxyethyl phosphate (hp). 4-thiouracil and 5- bromouracil substitutions can also be made. Parrish et al. 2000 Molecular Cell 6 : 1077-1087. [00528] Those skilled in the art will appreciate that it is possible to synthesize and modify the siRNA as desired, using any conventional method known in the art (see Henschel et al. 2004 DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Research 32 (Web Server Issue): W 113-W120). In addition, if the RNAi agent is a shRNA, it will be apparent to those skilled in the art that there are a variety of regulatory sequences (for example, constitutive or inducible promoters, tissue- specific promoters or functional fragments thereof, etc.) which are useful for shRNA expression construct/vector.

[00529] There are several examples in the art describing sugar, base, phosphate and backbone modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyl, 2'-0-allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren 1992 TIBS. 17: 34; Usman et al. 1994

Nucleic Acids Symp. Ser. 3 1: 163; Burgin et al. 1996 Biochemistry 35: 14090). Sugar modification of nucleic acid molecules are extensively described in the art.

[00530] In various aspects, the RNAi agent comprises a 2'-modification selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'- O-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), and 2'-0-N-methylacetamido (2'-0-NMA). [00531] Additional modifications and conjugations of RNAi agents have been described. Soutschek et al. 2004 Nature 432: 173-178 presented conjugation of cholesterol to the 3'-end of the sense strand of a siRNA molecule by means of a pyrrolidine linker, thereby generating a covalent and irreversible conjugate. Chemical modifications (including conjugation with other molecules) of RNAi agents may also be made to improve the in vivo pharmacokinetic retention time and efficiency.

[00532] In various aspects, the RNAi agent to Hepcidin comprises at least one 5'-uridine- adenine-3' (5'-ua-3') dinucleotide, wherein the uridine is a 2'-modified nucleotide; at least one 5'- uridine-guanine-3' (5'-ug-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; at least one 5'-cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; and/or at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide. In certain aspects, the RNAi agent can comprise a non-natural nucleobase, wherein the non-natural nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a particular aspect, the non-natural nucleobase is difluorotolyl. In certain aspects, only one of the two oligonucleotide strands contains a non-natural nucleobase. In certain aspects, both of the oligonucleotide strands contain a non-natural nucleobase. [00533] In another aspect, the RNAi comprises a gap or contains mismatch comprising an abasic nucleotide. [00534] In another aspect, the RNAi agent has a single-stranded nick (e.g., a break or missing bond in the backbone). In various aspects, a single-stranded nick can be in either the sense or anti-sense strand, or both. [00535] This nick can be, for example, in the sense strand, producing a small internally segmented interfering RNA, or sisiRNA, which may have less off-target effects than the corresponding RNAi agent without a nick. See, for example, WO 2007/107162 to Wengels and Kjems. [00536] The antisense nucleic acid or RNAi agent can also have an alternative backbone such as locked nucleic acids (LNA), Morpholinos, peptidic nucleic acids (PNA), threose nucleic acid (TNA), or glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2 ' -fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), or unlocked nucleic acid (UNA), and/or it can be labeled (e.g., radiolabeled or otherwise tagged). FANA are described in Dowler et al. 2006 Nucl. Acids Res. 34: 1669-1675. [00537] One or both strands can comprise an alternative backbone.

[00538] In yet another aspect, the RNAi agent employed by the methods of the present disclosure can include an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. Gaultier et al. 1987 Nucleic Acids. Res. 15: 6625-6641 . [00539] The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. 1987 Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. 1987 FEBS Lett. 215: 327-330). [00540] Other modifications and/or other changes can be made to the RNAi agent. A portion of the RNAi agent can be double-stranded DNA, while another portion is double- stranded RNA, forming a DNA-RNA chimera (See, for example, Yamato et al. 201 1. Cancer Gene Ther. 18: 587-597). Mismatches between the guide and passenger stand can also be introduced, though some positions may be better suited than others (See, for example, U.S. Patent App. No. 2009/0209626 to Khvorova). The passenger strand can also be shortened, to as short as 15 or 16 nt, while the guide strand remains 19 nt or longer (See, for example, Sun et al. 2008 Nature Biotech. 26: 1379-1382; and Chu and Rana 2008 RNA 14: 1714-1719). This can increase incorporation of the guide strand into the RNA-induced Silence Complex (RISC), and decrease incorporation of the passenger strand, than reducing off-target effects. In some cases, the passenger strand may be more amenable to modification (e.g., single-stranded nicking, nucleotide modifications, and shortening) than the guide strand. [00541] These and many other modifications can be made once a functional guide strand is identified.

[00542] Pharmaceutical Compositions of RNAi Agents [00543] As used here, a "pharmaceutical composition" comprises a pharmaceutically effective amount of one or more Hepcidin RNAi agent, a pharmaceutically acceptable carrier, and, optionally, an additional disease treatment which works synergistically with the RNAi agent. As used herein, "pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of a RNAi agent effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective where there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter. In this aspect, a therapeutically effective amount of a RNAi agent targeting Hepcidin can reduce Hepcidin protein levels by at least 10%. In additional aspects, a given clinical treatment is considered effective where there is at least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction in a measurable parameter associated with a disease or disorder, and the therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 15, 20, 25, 30, 35, 40, 45,

50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction, respectively, in that parameter. [00544] The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, lipid nanoparticles, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. Any appropriate phamaceutical carrier known in the art can be used in conjunction with the RNAi agents disclosed herein.

[00545] Pharmaceutical Composition Comprising a RNAi Agent to Hepcidin [00546] Additional components of a pharmaceutical composition comprising a RNAi Agent to Hepcidin are contemplated to aid in delivery, stability, efficacy, or reduction of immunogenicity. [00547] Liposomes have been used previously for drug delivery (e.g., delivery of a chemotherapeutic). Liposomes (e.g., cationic liposomes) are described in PCT publications WO02/100435A1, WO03/015757A1, WO04029213A2; and WO/201 1/076807; U.S. Pat. Nos. 5,962,016; 5,030,453; and 6,680,068; and U.S. Patent Application 2004/0208921 . A process of making liposomes is also described in WO04/002453A1 . Furthermore, neutral lipids have been incorporated into cationic liposomes (e.g., Farhood et al. 1995), as well as PEGylated lipids. [00548] Cationic liposomes have been used to deliver RNAi agent to various cell types (Sioud and Sorensen 2003; U.S. Patent Application 2004/0204377; Duxbury et al., 2004; Donze and Picard, 2002). [00549] Use of neutral liposomes disclosed in Miller et al. 1998, and U.S. Patent Application 2003/0012812. [00550] As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an RNAi or a plasmid from which an RNAi is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. [00551] Chemical transfection using lipid-based, amine-based and polymer-based techniques is disclosed in products from Ambion Inc., Austin, Tex.; and Novagen, EMD Biosciences, Inc, an Affiliate of Merck KGaA, Darmstadt, Germany); Ovcharenko D (2003) "Efficient delivery of siRNAs to human primary cells." Ambion TechNotes 10 (5): 15-16). Additionally, Song et al. (Nat Med. published online (Feb I 0, 2003) doi: 10.1038/nm828) and others [Caplen et al. 2001 Proc. Natl. Acad. Sci. (USA), 98: 9742-9747; and McCaffrey et al. Nature 414: 34-39] disclose that liver cells can be efficiently transfected by injection of the siRNA into a mammal's circulatory system. [00552] A variety of molecules have been used for cell-specific RNAi agent delivery. See, for example, WO/201 1/076807. For example, the nucleic acid-condensing property of protamine has been combined with specific antibodies to deliver siRNAs. Song et al. 2005 Nat Biotech. 23: 709-717. The self-assembly PEGylated polycation polyethylenimine (PEI) has also been used to condense and protect siRNAs. Schiffelers et al. 2004 Nucl. Acids Res. 32: el49, 141-1 10. [00553] The RNAi agents of the present disclosure can be delivered via, for example, Lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles; double-stranded RNA binding motifs (dsRBMs); or via modification of the RNAi agent (e.g., covalent attachment to the dsRNA) or by any method known in the art for delivery of a RNAi agent comprising nucleic acids. [00554] Lipid nanoparticles (LNP) are self-assembling cationic lipid based systems. These can comprise, for example, a neutral lipid (the liposome base); a cationic lipid (for siRNA loading); cholesterol (for stabilizing the liposomes); and PEG-lipid (for stabilizing the formulation, charge shielding and extended circulation in the bloodstream). [00555] The cationic lipid can comprise, for example, a headgroup, a linker, a tail and a cholesterol tail. The LNP can have, for example, good delivery to the diseased area, extended circulation in the blood, small particles (e.g., less than 100 nm), and stability in the microenvironment of the diseased area (which may have low pH and/or be hypoxic).

[00556] Neutral liposomes (NL) are non-cationic lipid based particles. [00557] Polymer nanoparticles are self-assembling polymer-based particles. [00558] Double-stranded RNA binding motifs (dsRBMs) are self-assembling RNA binding proteins, which will need modifications.

[00559] Hepcidin RNAi agent compositions in a Lipid nanoparticles (LNP) comprising a neutral lipid; a cationic lipid; cholesterol; and PEG-lipid [00560] Lipid nanoparticles (LNP) are self-assembling cationic lipid based systems. These can comprise, for example, a neutral lipid (the liposome base); a cationic lipid (for siRNA loading); cholesterol (for stabilizing the liposomes); and PEG-lipid (for stabilizing the formulation, charge shielding and extended circulation in the bloodstream). A neutral lipid is, for example, the liposome base. A cationic lipid is, for example, for siRNA loading. Cholesterol is, for example, for stabilizing the liposomes. PEG-lipid is, for example, for stabilizing the formulation, charge shielding and extended circulation in the bloodstream.

[00561] Additional pharmaceutical compositions [00562] In various aspects, the RNAi agent to Hepcidin is packaged as a monotherapy into a delivery vehicle, or may be further ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin. [00563] The RNAi agents of the present disclosure can be prepared in a pharmaceutical composition comprising various components appropriate for the particular method of administration of the RNAi agent.

[00564] Particular specific aspects [00565] In a particular specific aspect, the present disclosure is a composition comprising one or more Hepcidin RNAi agents.

[00566] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00567] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 85, or modified or unmodified variants thereof. [00568] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 86, or modified or unmodified variants thereof. [00569] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 87, or modified or unmodified variants thereof. [00570] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 88, or modified or unmodified variants thereof. [00571] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 89, or modified or unmodified variants thereof. [00572] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 90, or modified or unmodified variants thereof.

[00573] Particular specific aspects [00574] In a particular specific aspect, the present disclosure is a composition comprising one or more Hepcidin RNAi agents.

[00575] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00576] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 85, and the sequence of the second strand is the sequence of SEQ ID NO: 9 1, or modified or unmodified variants thereof. [00577] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 86, and the sequence of the second strand is the sequence of SEQ ID NO: 92, or modified or unmodified variants thereof. [00578] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 87, and the sequence of the second strand is the sequence of SEQ ID NO: 93, or modified or unmodified variants thereof. [00579] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 88, and the sequence of the second strand is the sequence of SEQ ID NO: 94, or modified or unmodified variants thereof. [00580] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 89, and the sequence of the second strand is the sequence of SEQ ID NO: 95, or modified or unmodified variants thereof. [00581] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 90, and the sequence of the second strand is the sequence of SEQ ID NO: 96, or modified or unmodified variants thereof.

[00582] Additional particular specific aspects [00583] In a particular specific aspect, the present disclosure is a composition comprising one or more Hepcidin RNAi agents.

[00584] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00585] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 85, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00586] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 86, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00587] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 87, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00588] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 88, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00589] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 89, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00590] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 90, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof.

[00591] Additional particular specific aspects [00592] In various aspects, the disclosure comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of any one or more RNAi agent disclosed herein, or modified or unmodified variants thereof.

[00593] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00594] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 85, or modified or unmodified variants thereof. [00595] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 86, or modified or unmodified variants thereof. [00596] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 87, or modified or unmodified variants thereof. [00597] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 88, or modified or unmodified variants thereof. [00598] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 89, or modified or unmodified variants thereof. [00599] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 90, or modified or unmodified variants thereof.

[00600] Additional particular specific aspects [00601] In various aspects, the disclosure comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from a first strand, or modified or unmodified variants thereof.

[00602] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00603] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 85, or modified or unmodified variants thereof. [00604] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 86, or modified or unmodified variants thereof. [00605] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 87, or modified or unmodified variants thereof. [00606] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 88, or modified or unmodified variants thereof. [00607] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 89, or modified or unmodified variants thereof. [00608] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 90, or modified or unmodified variants thereof.

[00609] Additional Particular specific aspects [00610] In a particular specific aspect, the present disclosure is a composition comprising one or more Hepcidin RNAi agents.

[00611] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00612] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 85, and/or the sequence of the second strand is the sequence of SEQ ID NO: 9 1, or modified or unmodified variants thereof. [00613] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 86, and/or the sequence of the second strand is the sequence of SEQ ID NO: 92, or modified or unmodified variants thereof. [00614] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 87, and/or the sequence of the second strand is the sequence of SEQ ID NO: 93, or modified or unmodified variants thereof. [00615] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 88, and/or the sequence of the second strand is the sequence of SEQ ID NO: 94, or modified or unmodified variants thereof. [00616] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 89, and/or the sequence of the second strand is the sequence of SEQ ID NO: 95, or modified or unmodified variants thereof. [00617] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand is the sequence of SEQ ID NO: 90, and/or the sequence of the second strand is the sequence of SEQ ID NO: 96, or modified or unmodified variants thereof.

[00618] Additional Particular specific aspects [00619] In a particular specific aspect, the present disclosure is a composition comprising one or more Hepcidin RNAi agents.

[00620] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00621] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 85, and/or the sequence of the second strand comprises the sequence of SEQ ID NO: 91, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00622] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 86, and/or the sequence of the second strand comprises the sequence of SEQ ID NO: 92, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00623] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 87, and/or the sequence of the second strand comprises the sequence of SEQ ID NO: 93, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00624] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 88, and/or the sequence of the second strand comprises the sequence of SEQ ID NO: 94, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00625] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 89, and/or the sequence of the second strand comprises the sequence of SEQ ID NO: 95, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00626] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises the sequence of SEQ ID NO: 90, and/or the sequence of the second strand comprises the sequence of SEQ ID NO: 96, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof.

[00627] Additional particular specific aspects [00628] In various aspects, the disclosure comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of a first strand, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides of the second strand of any any one or more RNAi agent disclosed herein.

[00629] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00630] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 85, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 9 1, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00631] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 86, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 92, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00632] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 87, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 93, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00633] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 88, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 94, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00634] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 89, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 95, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00635] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 90, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides of the sequence of SEQ ID NO: 96, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof.

[00636] Additional particular specific aspects

[00637] In various aspects, the disclosure comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from a first strand, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the second strand of any any one or more RNAi agent disclosed herein. [00638] In various aspects, the disclosure encompasses a composition comprising any one or more of the following: [00639] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 85, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from the sequence of SEQ ID NO: 9 1, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00640] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 86, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from the sequence of SEQ ID NO: 92, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00641] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 87, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from the sequence of SEQ ID NO: 93, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00642] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 88, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from the sequence of SEQ ID NO: 94, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00643] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 89, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from the sequence of SEQ ID NO: 95, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof. [00644] A RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of SEQ ID NO: 90, and/or the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from the sequence of SEQ ID NO: 96, wherein the length of the first and second strand are each no more than about 30 nt, or modified or unmodified variants thereof.

[00645] Additional particular aspects [00646] In various aspects, the disclosure comprises a RNAi agent comprising a sense and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from the antisense strand of any RNAi agent disclosed herein, or modified or unmodified variants thereof, wherein the antisense strand optionally further comprises 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9, or 10 or more nt (or any range thereof, e.g., 0-1, 1-2, 1-3, 1- 4 nt, etc.). [00647] Thus, in various aspects, the disclosure comprises a RNAi agent comprising a sense and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nt from any of: SEQ ID NOs: 85 to 90, or modified or unmodified variants thereof, wherein the antisense strand optionally further comprises 0, 1, 2, 3,

4, 5, 6, 7, 8, 9, or 10 or more nt (or any range thereof, e.g., 0-1 , 1-2, 1-3, 1-4 nt, etc.).

[00648] In one aspect, the disclosure comprises any one or more RNAi agent listed herein.

[00649] Additional particular specific aspects [00650] Other particular specific aspects include compositions comprising 1, 2 , 3, 4 , or more of these RNAi agents. Another aspect is a composition comprising any single RNAi agent, along with any other RNAi agents which overlap it. Another aspect comprises two, three, four or more Hepcidin RNAi agents which do not overlap and thus target different parts of the RNA molecule. When two or more RNAi agents are used, they can be administered simultaneously or sequentially. [00651] Another particular specific aspect comprises an RNAi agent, wherein the RNAi agent comprises a sense strand comprising at least 15 contiguous nucleotides (identical in sequence) to the sense strand of any of the listed RNAi agents, and an antisense strand comprising at least 15 contiguous nucleotides (identical in sequence) to the antisense strand of the same RNAi agent. In another aspect, the composition comprises one, two, three, four, or more such RNAi agents. [00652] In one aspect, the composition comprises an RNAi agent which comprises an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2 or 3 mismatches from the antisense strand of a RNAi agent described herein. [00653] In one aspect, the composition comprises an RNAi agent which comprises an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2 or 3 mismatches from the antisense strand of a RNAi agent described herein. [00654] In another aspect, the composition comprises an RNAi agent which comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 mismatches from the sense strand of one of the listed RNAi agents, and an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2 or 3 mismatches from the antisense strand of the same RNAi agent. [00655] A "mismatch" is defined herein as a difference between the base sequence or length when two sequences are maximally aligned and compared. As a non-limiting example, a mismatch is counted if a difference exists between the base at a particular location in one sequence and the base at the corresponding position in another sequence (e.g., between the sequence of a given RNAi agent and an RNAi agent listed herein). Thus, a mismatch is counted, for example, if a position in one sequence has a particular base (e.g., A), and the corresponding position on the other sequence has a different base (e.g., G, C or U). A mismatch is also counted, e.g., if a position in one sequence has a base (e.g., A), and the corresponding position on the other sequence has no base (e.g., that position is an abasic nucleotide which comprises a phosphate-sugar backbone but no base). A single-stranded nick in either sequence (or in the sense or antisense strand) is not counted as mismatch. Thus, as a non-limiting example, no mismatch would be counted if one sequence comprises the sequence A-G, but the other sequence comprises the sequence A-G with a single-stranded nick between the A and the G. A base modification is also not considered a mismatch. If one sequence comprises a C, and the other sequence comprises a modified C (e.g., with a 2'-modification) at the same position, no mismatch would be counted. Thus, modifications of a nucleotide other than replacement or alteration of the base would not constitute a mismatch. For example, no mismatch would occur between a nucleotide which is A , and a nucleotide which is A with a 5' modification and/or a 2'-modification. The key feature of a mismatch (base replacement) is that it would not be able to base-pair with the corresponding base on the opposite strand. In addition, terminal overhangs such as "UU" or "dTdT" are not counted when counting the number of mismatches; the terminal "UU" and "dTdT" overhangs are also not included when calculating

" 15 contiguous nucleotides." [00656] In these aspects, a mismatch is defined as a position wherein the base of one sequence does not match the base of the other sequence. [00657] In another aspect, the composition comprises 1, 2 , 3, 4 , or more such RNAi agents. [00658] In another aspect, the composition comprises an RNAi agent which comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2 or 3 mismatches from the sense strand of one of the listed RNAi agents, and an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2 or 3 mismatches from the antisense strand of the same RNAi agent

[00659] Variants of RNAi agents (e.g., comprising different modifications, caps, etc.) are disclosed herein, e.g., in the Tables. An unmodified and an example modified variant of various RNAi agents are provided. The disclosure thus encompasses groups of overlapping modified and/or unmodified RNAi agents. More aspects are provided herein, and are included in the scope of each RNAi agents of the disclosure.

[00660] Other modifications known to one skilled in the art are contemplated as being encompassed within the invention. Exemplary modifications include, but are not limited to, the presence of gaps or mismatches between the base pairs in the sense and antisense strands, the presence of nicks or breaks in the internucleoside linkages in the sense strand, and the like.

[00661] PHARMACEUTICAL COMPOSITIONS [00662] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. [00663] Oral administration of the compositions of the invention include all standard techniques for administering substances directly to the stomach or gut, most importantly by patient controlled swallowing of the dosage form, but also by other mechanical and assisted means of such delivery. [00664] Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above- indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about

1 mg to about 500 mg of an active ingredient. It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy. [00665] Therapeutic effect of the therapeutic agents of the invention may be enhanced by combination with other agents. Typically such other agents will include agents known for use in treating similar diseases, such as angiogenic disorders. [00666] The RNAi agents of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, intraperitoneal, or intrathecal injection, or infusion techniques and the like. Where two or more different RNAi agents are administered, each may be administered separately or co-administered. Where each is adminitered separately, the method and/or site of adminstration may be the same or different, e.g., both RNAi agents may be administered intraveneously or subcutaneously, or a first RNAi agent may be administered intraveneously with a second Rai agent administered subcutaneously, etc.

[00667] In various embodiments, the disclosure encompasses a composition or pharmaceutical composition comprising a RNAi agent, wherein one or both strands comprises a 3' end cap, the composition further comprising a helper lipid, a neutral lipid, and/or a stealth lipid.

[00668] In one particular specific embodiment, the present disclosure relates to a method of treating a target gene-related disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the first and/or second strand comprise a

3' end cap selected from the 3' end caps listed in Table 2 . In one particular specific embodiment, the present disclosure relates to a method of inhibiting the expression of target gene in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition comprising a RNAi agent of the present disclosure.

[00669] In one embodiment of the method, the composition further comprises a pharmaceutically effective formulation. [00670] Various particular specific embodiments of these embodiments are described below.

[00671] In one embodiment, the method further comprises the administration of an additional treatment. In one embodiment, the additional treatment is a therapeutically effective amount of a composition.

[00672] In one embodiment, the additional treatment is a method (or procedure). [00673] In one embodiment, the additional treatment and the RNAi agent can be administered in any order, or can be administered simultaneously. [00674] In one embodiment, the method further comprises the step of administering an additional treatment for the disease. [00675] In one embodiment, the method further comprises the step of administering an additional treatment or therapy selected from the list of an additional antagonist to a target gene-related disease. [00676] In one embodiment, the composition comprises a second RNAi agent to target gene. In various embodiments, the second RNAi agent is physically separate from the first, or the two are physically connected (e.g., covalently linked or otherwise conjugated). [00677] [00678] Other embodiments [00679] Various particular specific embodiments of this disclosure are described below. [00680] In one embodiment, the disclosure pertains to a composition according to any of the embodiments described herein, for use in a method of treating a target gene-related disease in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a composition according to any of the claims. [00681] One embodiment of the disclosure is the use of a composition according to any of these embodiments, in the manufacture of a medicament for treatment of an target gene-related disease. [00682] In one embodiment, the disclosure pertains to the composition of any of the above embodiments, for use in the treatment of an target gene-related disease.

[00683] Additional Definitions [00684] Unless defined otherwise, the technical and scientific terms used herein have the same meaning as that usually understood by a specialist familiar with the field to which the present disclosure belongs. [00685] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein. [00686] Claims to the present disclosure are non-limiting and are provided below. [00687] Although particular embodiments and claims have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, or the scope of subject matter of claims of any corresponding future application. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the present disclosure without departing from the spirit and scope of the present disclosure as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein or known in the art. Other aspects, advantages, and modifications considered to be within the scope of the following claims. Redrafting of claim scope in later-filed corresponding applications may be due to limitations by the patent laws of various countries and should not be interpreted as giving up subject matter of the claims. [00688] Various additional formulations and obvious variants of the described 3' end caps can be devised by those of ordinary skill in the art. Non-limiting example RNAi agents wherein one or both strands comprises a 3' end cap are described in the Examples below, which do not limit the scope of the present disclosure as described in the claims.

EXAMPLES

EXAMPLE 1. In vitro potency of Hepcidin RNAi agents Potency of HAMP siRNA - PAZ Ligand Conjugates is studied. Hepcidin mRNA down regulation in HuH-7 cells is studied at 3 doses.

Two test sequences: hs_HAMP_400 and 402

Parent stem format: A106S42 (2'-OMe chemistry

19 PAZ ligands on guide (antisense) strand +/- ribitol spacer, +/- MOE clamp (wherein the MOE clamp is a 2'-MOE modification on each of the two last base-pairing nucleotides on each strand counting from 5' to 3').

In vitro dose-response in Huh-7 cells is determined.

Results are shown in Figs. 2A and 2B and 7 and in Table 5, below.

Figs. 2A and 2B show the in vitro RNA interference or KD (knockdown) mediated by various RNAi agents comprising a 3' end cap: BP (biphenyl), C6, X027, X038, X050, X051, X052, X058, X059, X060, X061 , X062, X063, X064, X065, X066, X067, X068, and X069 on the guide (antisense) strand. Two sequences were tested (hs_HAMP_400 and _402), where 400 is a sequence beginning at position 400 of the human HAMP (Hepcidin) gene and 402 is an overlapping sequence beginning at position 402 These were tested with (Rib) and without (-) a ribitol spacer; and with (MOE) and without (-) a 2'-MOE clamp (as diagrammed in Fig. 2A and 2B). Various hsJHAMP 400 and 402 are depicted in Figs. 3A (Guide or antisense strand) and 3B (corresponding Sense strand).

The data are provided in Figs. 2A and 2B.

Additional data is provided in Table 7, below. This Table indicates the Nickname ("Oligo Identifier") for the 3' end cap, and the DMT, Succinate and Carboxylate variants thereof; the Carboxylate Kd; the KD (knockdown) mediated by a Hepcidin RNAi agent comprising the 3' end cap (format: S402 + ribitol + MOE clamp) at 5 nM in vitro; and the approximate (approx.) IC50

Example 2a. Synthesis of siRNA conjugated with X1062

To AICI 3 (7.88 g, 59.1 mmol) in Toluene (200 ml) was added aniline (5g, 53.7 mmol, 4.59 ml, in 50 ml Toluene) dropwise under N2. 3-bormobenzonitrile (29.3 g, 161 mmol) was added to the above mixture subsequently. The resulting mixture was stirred at RT for 1h, then heated at 110°C for 6 hrs. The reaction mixture was cooled to RT, to which aq. HCI ( 1 M, 3 ml) was added. The solution was then heated at 80°C for 1h. The solution was cooled to RT, and the organic layer and water layer were separated. The water layer was extracted with ethyl acetate (3 x 100ml_). The combined organic layers were washed with water and brine, and dried over sodium sulfate. The organic solvent was then removed under vacuum. The crude product was purified by silica chromatography with 0-40% ethyl acetate/heptane to give 1 (4.31 g, 15.6 mmol) in 29% yield. ESI MS (m/z, MH+): 278.1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 6.16 (br. s., 2 H) 6.64 (t, J=7.53 Hz, 1 H) 6.76 (d, J=8.53 Hz, 1 H) 7.29 - 7.49 (m, 3 H) 7.56 (d, J=7.53 Hz, 1 H) 7.67 (d, J=8.03 Hz, 1 H) 7.74 - 7.84 (m, 1 H)

A mixture of 1 ( 1 .02 g, 3.69 mmol) and ethyl 4-chloro-4-oxobutanoate (0.669 g, 4.06 mmol) in DCM (60 ml) was heated at 60°C for 1h. The reaction mixture was cooled and quenched with aq. 1 M NaOH (15 ml). The organic layer and water layer were separated. The water layer was extracted with dichloromethane (3 x 50ml). The combined organic layers were washed with water and brine, and dried over sodium sulfate. The organic solvent was then removed under vacuum to give 2 ( 1 .44 g, 3.56 mmol) in 96% yield. ESI MS (m/z, MH+): 406.2. H NMR (400 MHz, Chloroform-d) δ 10.88 (s, 1H), 8.65 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 1.8 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.65 - 7.51 (m, 3H), 7.39 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.7 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 2.77 (dt, J = 10.3, 5.2 Hz, 4H), 1.27 (t, J = 7.1 Hz, 4H). A mixture of 2 ( 1 .44 g, 3.56 mmol) and sodium hydride ( 1 .425g, 35.6 mmol) in ethanol (20 ml) was heated at 80°C for 2h. The reaction mixture was cooled to RT and quenched with water (5 ml) then neutralized with aq. 1 M HCI (2 ml). The resulting solution was extracted with ethyl acetate (3 x 50ml_). The combined organic layers were washed with water and brine, and dried over sodium sulfate. The organic solvent was then removed under vacuum to give 3 ( 1 .2 g, 3.63 mmol) in 94% yield. ESI MS (m/z, MH+): 360.2. H NMR (400 MHz, Chloroform-d) δ 11.71 - 11.63 (m, 1H), 11.55 - 11.42 (m, 3H), 11.37 (d, J = 8.3 Hz, 1H), 11.30 - 11.22 (m, 1H), 11.12 (td, J = 7.6, 7.0, 1.2 Hz, 1H), 11.00 (dd, J = 8.2, 1.4 Hz, 1H), 7.33 (d, J = 3.5 Hz, 2H), 4.87 - 4.82 (m, 2H).

A solution of 3 ( 1 .2 g, 1.89 mmol) in POCI3 (15 ml) was stirred at RT for 2h. POCI3 was removed under vacuum, the resulting residue was quenched with ethanol (50 ml). The solution was stirred at RT for 2h, then ethanol was removed under vacuum. To the residue was added dichloromethane (50 ml) and aq. 1 M NaOH (20ml). Organic layer and water layer were separated. The water layer was extracted with dichloromethane (2 x 50 ml). The combined organic layers were washed with water, brine and dried over sodium sulfate. The organic solvent was removed under vacuum to give 4 ( 1 .76 mg, 4.56 mmol) in 136% yield. ESI MS (m/z, MH+): 388.2. H NMR (400 MHz, Chloroform-d) δ 7.65 (dt, J = 8.2, 1.4 Hz, 1H), 7.54 - 7.46 (m, 2H), 7.45 - 7.37 (m, 2H), 7.27 (dt, J = 7.8, 1.5 Hz, 1H), 7.17 - 7.04 (m, 2H), 4.18 - 4.16 (m, 2H), 3.50 (d, J = 1.5 Hz, 2H), 1.27 (h, J = 3.7 Hz, 3H). A solution of 4 ( 1 .76 g, 4.56 mmol) in POCI3 (10 ml) was heated at 110°C for 12h. POCI3 was removed under vacuum. To the residue was added dichloromethane (50 ml) and aq. 1 M NaOH (20ml). The organic layer and water layer were separated. The water layer was extracted with dichloromethane (2 x 50 ml). The combined organic layers were washed with water, brine and dried over sodium sulfate. The organic solvent was removed under vacuum to give 5 (440 mg, 1.09 mmol) in 32.5% yield. ESI MS (m/z, MH+): 406.0. H NMR (400 MHz, Chloroform-d) δ 8.14 - 8.05 (m, 1H), 7.76 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.69 (ddd, J = 8.0, 1.9, 1.0 Hz, 1H), 7.53 - 7.41 (m, 3H), 7.36 (dd, J = 8.3, 1.3 Hz, 1H), 7.26 (dt, J = 7.7, 1.3 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.72 (s, 2H), 1.27 (t, J = 7.1 Hz, 3H).

A mixture of 5 (50 mg, 0.124 mmol), Zinc power (40.4 mg, 0.618 mmol) in TFA ( 1 ml) was heated at 40°C for 12h. The reaction mixture was quenched with NaOH (1M, 1 ml). The organic layer was extracted with dichloromethane (2 x 10 ml). The combined organic layers were washed with water, brine and dried over sodium sulfate. The organic solvent was removed under vacuum. The crude product was purified by silica flash chromotograph with elute 0-50% EtOAc/Heptane to give 6 (39 mg, 0.105 mmol) in 85% yield. ESI MS (m/z, MH+): 372.1 . H NMR (400 MHz, CHLOROFORM-d) ppm 1.24 (t, J=7.15 Hz, 3 H) 3.63 (s, 2 H) 4.02 - 4.27 (m, 2 H) 7.12 - 7.33 (m, 1 H) 7.37 - 7.61 (m, 4 H) 7.61 - 7.71 (m, 1 H) 7.75 (t, J=1 .51 Hz, 1 H) 8.21 (d, J=8.53 Hz, 1 H) 8.94 (s, 1 H). A mixture of 4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ethyl)-1,3-dioxolan-2-one) (255 mg, 1.05 mmol), 6 (39 mg, 0.105 mmol), (Brettphos)paddadium(ll) phenethylamine chloride (4.21 mg, 5.27 umol) and 1M aqueous K3P0 4 (421 uL, 0.421 mmol) in 2-Me THF (500 uL) and DMF (500uL) was heated at 80°C for 12h. The reaction mixture was filtered to remove insoluable material. The organic solvent was removed under vacuum. The crude was purified by HPLC with 5% TFA in 5-95% acetonitrile/water to give 7 (15 mg, 0.04 mmol) in 37.5% yield. ESI MS (m/z, MH+): 380.4. H NMR (400 MHz, CHLOROFORM-d) ppm 1.22 (td, J=7.15, 2.26 Hz, 3 H) 1.73 - 1.92 (m, 2 H) 2.72 - 2.98 (m, 2 H) 3.49 (dd, J=1 1.04, 7.53 Hz, 1 H) 3.56 - 3.84 (m, 4 H) 4.04 - 4.20 (m, 2 H) 7.15 (d, J=7.53 Hz, 1 H) 7.13 (d, J=8.78 Hz, 1 H) 7.37 (d, J=7.53 Hz, 1 H) 7.43 - 7.56 (m, 3 H) 7.74 (br. s., 1 H) 8.22 (br. s., 1 H) 8.94 (br. s., 1 H).

A mixture of 7 (15 mg, 0.04 mmol) and aq. 1 M LiOH (87 uL ml, 0.087 mmol) in dioxane (200 ul) was stirred at RT for 12hrs. The organic solvent was removed under vacuum to give 8 (14.17 mg, 0.04 mmol) in 100% yield as lithium salt. ESI MS (m/z, MH+): 352.4. H NMR (400 MHz, METHANOL-^) δ ppm 1.62 - 1.80 (m, 1 H) 1.80 - 1.98 (m, 1 H) 2.78 (ddd, J=13.33, 6.76, 3.16 Hz, 1 H) 2.84 - 2.99 (m, 1 H) 3.46 - 3.55 (m, 3 H) 3.55 - 3.68 (m, 3 H) 6.98 - 7.25 (m, 2 H) 7.31 - 7.59 (m, 4 H) 7.74 (ddd, J=8.40, 6.38, 1.89 Hz, 1 H) 8.06 (d, J=8.34 Hz, 1 H) 8.86 (s, 1 H). 8 9

A mixture of 8 (14 mg, 0.039 mmol), TBDMSCI and imidazole (43.6 mg, 0.641 mmol) in DMF (4 ml) was stirred at RT for 24 hrs. The reaction mixture was quenched with water ( 1 ml). The organic layer was extracted with ethylestate (3 x 5 ml). The combined organic layers were washed with water, brine and dried over sodium sulfate. The organic solvent was removed under vacuum. The crude product was purified by silica flash chromotograph with elute 0-50% EtOAc/Heptane to give 9 (15.9 mg, 0.025 mmol) in 63.2% yield. ESI MS (m/z, MH+): 580.6. H NMR (400 MHz, METHANOL-^) δ ppm -0.12 - 0.13 (m, 8 H) 0.69 - 0.98 (m, 12 H) 1.17 - 1.36 (m, 5 H) 2.60 - 2.90 (m, 2 H) 3.38 - 3.62 (m, 6 H) 3.73 (d, J=4.55 Hz, 1 H) 7.02 - 7.15 (m, 2 H) 7.24 - 7.38 (m, 1 H) 7.39 - 7.47 (m, 3 H) 7.64 - 7.74 (m, 1 H) 7.98 - 8.06 (m, 1 H) 8.68 - 8.89 (m, 1 H).

A mixture of /V-hydroxysuccinimide (2.49 mg, 0.022 mmol), 9 (6.26 mg, 0.01 1 mmol), and DIC (2.74 mg, 0.022 mmol) in DMSO (200 uL) was stirred at RT for 12hrs. 10 uL of reaction mixture was diluted with 190 uL DMSO. To the resulting solution was added a freshly ss-siRNA-(CH 2 )3- NH2 solution (2mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reaction mixture was vortexed and sat at RT for 2h. The crude product was purified by HPLC with 10-40% 100 mM triethylammonium acetate in acetonitrile/water to afford 10. TOF MS (ES ): 6478. Example 2b. Synthesis of siRNA conjugated with X1063

Scheme 2 : Overview of the synthesis of 11.

A mixture of /V-hydroxysuccinimide (2.49 mg, 0.022 mmol), 9 (6.26 mg, 0.01 1 mmol), and DIC (2.73 mg, 0.022 mmol) in DMSO (200 uL) was stirred at RT for 12h. 10 uL of reaction mixture was diluted with 190 uL DMSO. To the resulting solution was added a freshly ss-siRNA-(CH 2 )4- NH2 solution (2mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reaction mixture was vortexed and sat at RT for 2h. The crude product was purified by HPLC with 10-40% 100 mM triethylammonium acetate in acetonitrile/water to afford 1 1 . TOF MS (ES ): 6492. Example 2c. Synthesis of siRNA conjugated with X1064

Scheme 3: Overview of the synthesis of 12.

A mixture of /V-hydroxysuccinimide (2.48 mg, 0.022 mmol), 9 (6.26 mg, 0.01 1 mmol), and DIC (2.72 mg, 0.022 mmol) in DMSO (200 uL) was stirred at RT for 12h. 10 uL of reaction mixture was diluted with 190 uL DMSO. To the resulting solution was added a freshly ss-siRNA-(CH 2 )5- NH2 solution (2mg, 0.325 umol in 80 uL PBS 8.5 buffer). The reaction mixture was vortexed and sat at RT for 2h. The crude product was purified by HPLC with 10-40% 100 mM triethylammonium acetate in acetonitrile/water to afford 12. TOF MS (ES ): 6506.

TABLE 7. These data thus show that that the human Hepcidin RNAi agents designated 400 and 402 were efficacious in the 18-mer format. Any of various 3' end cap have been used to make efficacious human Hepcidin RNAi agents, including BP (biphenyl), C6, X027, X038, X050, X051 , X052, X058, X059, X060, X061, X062, X063, X064, X065, X066, X067, X068, and X069.

EXAMPLE 3. Overlapping Hepcidin RNAi agents

[00689] Some of the siRNAs listed above overlap each other in sequence. [00690] For example, each of the 6 lead candidate sequences shares the common technical feature of UCCAAGACCUAU (SEQ ID NO: 168) in the antisense (AS) strand, and AUAGGUCUUGGA (SEQ ID NO: 169) in the sense (S) strand. Thus, the disclosure encompasses any group of 2 , 3, 4, 5 or all of the 6 lead candidates (e.g., 400 and 402; 400, 401, 402 and 403; 397 and 398; 397 and 400; 398 and 401 ; 398, 400, 402 and 403; etc.) [00691] It is also noted that 400 and 402 share the common technical feature of UAUUCCAAGACCUAUG (SEQ ID NO: 170) in the antisense strand and CAUAGGUCUUGGAAUA (SEQ ID NO: 171) in the sense strand. Thus, this disclosure encompasses the pair of human Hepcidin RNAi agents 400 and 402. [00692] Thus, in various aspects, the disclosure pertains to a group of overlapping RNAi agents which comprise or consist of; or which comprise a sense strand or and anti-sense strand of; or which comprise a sense strand and/or and anti-sense strand comprising at least 15 contiguous nt with 0, 1, 2 or 3 mismatches from the sense and/or anti-sense strand of: any subset of 2-6 of the lead candidates; and modifications and variants thereof.

[00693] EXAMPLE 4. Efficacy of additional 3' end caps [00694] The 3' end caps X1062, X1063 and X1064 were each found to be efficacious when used on RNAi agents. For example, these were effective on HuR siRNAs, wherein the HuR siRNAs were 18-mers as described herein, wherein the 3' end of each strand terminates in a phosphate and further comprises, in 5' to 3' direction, a spacer which is ribitol, a second phosphate, and a 3' end cap which is X1062, X1063 or X1064. Huh7 cells were transfected with siRNAs using RNAi Max transfection reagent; 24-well plates were seeded with 40,000 cells per well; reverse transfection was performed with 1 nM siRNA per well, and cells were incubated for about 18 hours. Transfection medium was replaced (without siRNA), and cells were incubated for an additional 2 days before RNA isolation or split for seeding. Cells were split on days 3 and 7 post transfection for duration time points. RNA was isolated at days 3, 7 and 10 post-transfection for HuR mRNA analysis. HuR siRNAs with 3' end caps which were X1062 demonstrated efficacy (knockdown) of 89.0, 77.9 and 32.7% after 3, 7 or 10 days. HuR siRNAs with 3' end caps which were X1063 and X1064 showed 89.6, 8 1.5, and 43.7%; and

67.0, 30.9 and 0.0%, respectively, after 3, 7 and 10 days.

[00695] Unless defined otherwise, the technical and scientific terms used herein have the same meaning as that usually understood by a specialist familiar with the field to which the disclosure belongs. [00696] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein. Unless indicated otherwise, each of the references cited herein is incorporated in its entirety by reference. [00697] Claims to the invention are non-limiting and are provided below. [00698] Although particular aspects and claims have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, or the scope of subject matter of claims of any corresponding future application. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the disclosure without departing from the spirit and scope of the disclosure as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the aspects described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific aspects of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Redrafting of claim scope in later filed corresponding applications may be due to limitations by the patent laws of various countries and should not be interpreted as giving up subject matter of the claims. We claim:

1. A composition comprising a Hepcidin RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2 , or 3 nucleotides from the antisense strand of an RNAi agent to Hepcidin provided in any of Tables 1 to 4 and 6, wherein the sense and/or the antisense strand is less than about 49 nucleotides in length.

2 . The composition of claim 1, wherein 3' end of the sense and/or antisense strand terminates in a phosphate or modified internucleoside linker.

3. The composition of claim 1, wherein 3' end of the sense and/or antisense strand terminates in a phosphate or modified internucleoside linker and further comprises a 3' end cap.

4 . The composition of claim 1, wherein the sense strand and the antisense strand form a duplex region about 15 to about 30 nucleotide pairs in length.

5. The composition of claim 1, wherein the antisense strand and the sense strand are both about 18 to about 23 nucleotides in length.

6. The composition of claim 1, wherein each strand of the RNAi agent is an 18-mer.

7. The composition of claim 1, wherein the RNAi agent comprises a modified sugar, a modified phosphorothioate linkage, or a 2'-modified nucleotide.

8. The composition of claim 1 or 6, wherein the 3' end of the sense and/or antisense strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer, a second phosphate or modified internucleoside linker, and a 3' end cap.

9. The composition of claim 8, wherein the spacer is selected from ribitol, 2'-deoxy-ribitol, diribitol, 2'-methoxyethoxy-ribitol, C3, C4, C5, C6, or 4-methoxybutane-1 ,3-diol.

10. The composition of claim 8, the modified internucleoside linker is selected from phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, an amide linker, and a compound of formula (I): (I), where R is selected from O , S , NH2, BH , C H , C C i o C C -io C _ C i o 3 3 -6 alkyl, 6- aryl, -6 alkoxy and 6 aryl-oxy, wherein 6 alkyl and 6- aryl are unsubstituted or optionally independently substituted with 1 to 3 groups independently selected 4 from halo, hydroxyl and NH2; and R is selected from O, S, NH, and CH2.

11. The composition of any of claims 2 or 8-10, wherein the 3' end cap is:

(a) A compound of formula la:

la

in which: X is the 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising the sense or antisense strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and optionally further comprises in 5' to 3' order: a spacer, and a second phosphate or modified internucleoside linker; Y is selected from CH and N;

m is selected from 0 and 1; p is selected from 1, 2 and 3;

R3 is selected from hydrogen, 2-(hydroxy-methyl)-benzyl, 3-(hydroxy-methyl)-benzyl and succinate, or is attached to a solid support; wherein the (CH -0-R moiety is 2 ) m 3 attached to the phenyl ring at position 3 or 4 ;

R4 is hydrogen; R5 is hydrogen; or R4 and R5, together with the phenyl rings to which R4 and R5 are attached, form 6H- benzo[c]chromene;

or

(b) A compound of formula lb: in which: X is the 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising the sense or antisense strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises in 5' to 3' order: a spacer, and a second phosphate or modified internucleoside linker; q is selected from 0, 1 and 2;

R6 is selected from phenyl which is unsubstituted or substituted with a group selected from benzoxy and 3,4-dihydroxybutyl;

R7 is selected from hydrogen and hydroxy-ethyl, wherein if R7 is hydroxy-ethyl, the hydroxyl can be optionally functionalized as succinate or attached to a solid support;

R8 is selected from hydrogen and methoxy; Y is selected from CH and N; and

Y2 is selected from N and CR9; wherein R9 is selected from hydrogen and methyl.

12. The composition of claim any of 3 or 8-10, wherein the 3' end cap is selected from any of: 171

ı 74 ı 75 In which: X is the 3' end of a Hepcidin RNAi agent strand, or the 3' end of a molecule comprising the first or second strand, wherein the 3' end of the strand terminates in a phosphate or modified internucleoside linker and further comprises in 5' to 3' order: a spacer, and a second phosphate or modified internucleoside linker; or

Wherein the 3' end cap is selected from any of: triethylene glycol, cyclohexyl, phenyl, lithochol (lithocholic acid), or adamantane.

13. The composition of claim 1, wherein the RNAi agent is ligated to one or more diagnostic compound, reporter group, cross-linking agent, nudease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.

14. The composition of claim 1, wherein the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 50% in Huh-7 tumors in nude mice. 15. The composition of claim 1, wherein the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 70% at a concentration of 10 nM in Huh-7 cells in vitro.

16. The composition of claim 1, wherein the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 80% at a concentration of 10 nM in Huh-7 cells in vitro.

17. The composition of claim 1, wherein the RNAi agent is capable of inhibiting expression of Hepcidin by at least about 90% at a concentration of 10 nM in Huh-7 cells in vitro.

18. The composition of claim 1, wherein the RNAi has an EC50 of no more than about 0.1 nM.

19. The composition of claim 1, wherein the RNAi has an EC50 of no more than about 0.01 nM.

20. The composition of claim 1, wherein the RNAi has an EC50 of no more than about 0.001 nM.

2 1. A method of treating a Hepcidin-related disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition according to claim 1.

22. The method of claim 21, wherein the Hepcidin-related disease is anemia.

23. The method of claim 21, wherein the Hepcidin-related disease is anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and hair loss.

24. The method of claim 21, wherein the method further comprises the step of administering an additional treatment.

25. The method of claim 24, wherein wherein administration of the composition comprising the RNAi agent and the additional treatment is simultaneous, concurrent, separate or sequential.

26. The method of claim 21, wherein the Hepcidin-related disease is a anemia. 27. The method of claim 2 1, where the Hepcidin-related disease is anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and hair loss.

28. The method of claim 21, wherein the method further comprises the step of administering an additional treatment.

29. The method of claim 21, wherein administration of the composition comprising the RNAi agent and the additional treatment is simultaneous, concurrent, separate or sequential

30. The method of claim 21, wherein the Hepcidin-related disease is anemia.

3 1. The method of claim 21, wherein the Hepcidin-related disease is anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and hair loss.

32. The method of claim 2 1, wherein the composition comprises a second RNAi agent to Hepcidin.

33. A method of inhibiting the expression of Hepcidin in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition of claim 1.

34. The composition according to claim 1, for use in a method of treating a Hepcidin-related disease in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a composition according to claim 1 or claim 2 1. 35. The composition according to claim 1, for use in a method of inhibiting the expression of

Hepcidin in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of a composition according to claim 1 or claim 2 1.

36. The use of a composition according to claim 1, in the manufacture of a medicament for treatment of an Hepcidin-related disease.

37. The use of claim 36, wherein the Hepcidin-related disease is anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al) (sometimes designated anemia of chronic disease or ACD), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and hair loss.

38. The composition of claim 1, for use in the treatment of an Hepcidin-related disease.

39. The composition of claim 38, wherein the Hepcidin-related disease is anemia, iron-deficient erythropoiesis, hypoferremia, impaired dietary iron uptake, iron sequestration, anemia of inflammation (Al) (sometimes designated anemia of chronic disease or ACD), atherosclerosis, diabetes, and multiple neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Friedrich's ataxia, heart failure, chronic kidney disease, cardiorenal-anemia syndrome, infection, blood loss, hemolysis, vitamin B12 or folate deficiency, hyperparathyroidism, hemoglobinopathies and malignancies, cancer, AIDS, surgery, runted growth, and hair loss.

40. A composition comprising an RNAi agent comprising a first strand and a second strand, wherein the first strand and/or second strand comprise at least 15 contiguous nucleotides from the first and second strand, respectively, of an RNAi agent to Hepcidin provided in any of Tables 1 to 4 and 6, wherein the first and/or second strand are less than about 49 nucleotides in length.

4 1. A composition comprising an RNAi agent comprising a first strand and a second strand, wherein the sequence of the first and/or second strand comprise the sequence of the first and/or second strand, respectively, of an RNAi agent to Hepcidin provided in any of Tables 1 to 4 and 6, wherein the first and/or second strand are less than about 49 nucleotides in length.

42. A composition comprising an RNAi agent comprising a first strand and a second strand, wherein the sequence of the first and/or second strand is the sequence of the first and/or second strand, respectively, of human Hepcidin RNAi agent 400.

43. A composition comprising an RNAi agent comprising a first strand and a second strand, wherein the sequence of the first and/or second strand is the sequence of the first and/or second strand, respectively, of human Hepcidin RNAi agent 402.

44. The composition of any of claims 42 or 43, wherein the 3' end of the first and/or second strand terminates in a phosphate or modified internucleoside linker and further comprises, in 5' to 3' order: a spacer, a second phosphate or modified internucleoside linker, and a 3' end cap.