Compositions and methods for inhibiting transmembrane serine protease 6 (TMPRSS6) expression

Abstract

Provided herein are oligonucleotides that inhibit the expression of TMPRSS6. Also provided are compositions comprising the oligonucleotides and uses thereof, in particular to uses relating to the treatment of diseases, disorders and / or conditions associated with hepcidin deficiency or inhibition.

Description

Background Art

[0001] Iron is a micronutrient and a biologically essential component of every living organism. It is required for adequate erythropoietic function, oxidative metabolism, cellular immune responses and many other cellular processes. In free form, iron participates in redox reactions leading to the formation of free radicals, oxidative stress, which can lead to organ damage. Living organisms have developed protein systems to transport free iron across cell membranes and biofluids and store it in a non-toxic and easily mobilized form to avoid iron toxicity. In the human body, iron is mainly present in the form of complexes bound to proteins such as heme compounds, heme enzymes or non-heme compounds. Iron absorption is carried out by enterocytes via divalent metal transporter 1 and occurs in the duodenum and upper jejunum. The iron is then transferred across the duodenal mucosa into the blood where it is transported by transferrin to cells or bone marrow for erythropoiesis. Since iron is required for many different cellular functions, a constant balance between iron uptake, transport, storage and utilization needs to be maintained to maintain iron homeostasis. Hepcidin is a circulating peptide hormone encoded by the HAMP gene that is secreted by the liver and plays a key role in maintaining iron homeostasis. Hepcidin expression is directly regulated by changes in iron intake, and its inhibition leads to elevated levels of bioavailable serum iron. Hepcidin blocks the influx of iron into the plasma by directly binding to ferroportin, the major iron exporter located at the cell membrane of duodenal enterocytes, which absorb iron from the diet, and macrophages, which recycle iron from aging erythrocytes. Hepcidin binding to ferroportin triggers its internalization and subsequent lysosomal degradation, thereby preventing iron export into the circulation. Hepatic hepcidin expression is regulated by three independent pathways, where inflammation (inflammatory cytokines) and high serum iron levels lead to hepcidin upregulation, while increased erythropoiesis leads to hepcidin downregulation, thereby promoting the high iron demand for erythrocyte production. In other words, when hepcidin levels are high, serum iron levels are reduced, which can lead to anemia. When hepcidin levels are low, as in disease states such as hemochromatosis, iron levels increase and overload may occur.

[0002] In pathological situations, chronic inhibition of hepcidin often results in primary iron overload, due to loss of function of plasma iron sensing signaling pathways leading to impaired expression (e.g., hereditary hemochromatosis). In conditions of increased but dysfunctional erythropoiesis (e.g., beta-thalassemia), hepcidin is downregulated, leading to secondary iron overload. The pathophysiological consequences of both primary and secondary iron overload are common, leading to accumulation of free plasma iron, which accumulates in organs and causes damage as the disease progresses.

[0003] TMPRSS6 (transmembrane protease, serine 6) encodes a type II serine protease and is primarily expressed in the liver. TMPRSS6 participates in transmembrane signaling pathways triggered by iron deficiency and inhibits multiple pathways that activate HAMP, the gene encoding hepcidin.

[0004] Studies have shown that in mouse models of intermediary β-thalassemia and hereditary hemochromatosis, upregulation of hepcidin improves abnormal erythropoiesis and prevents or limits iron overload (Gardenghi et al., J. Clin. Invest., 2010; Guo et al., J. Clin. Invest., 2013; Schmidt et al., Blood 2013; Casu et al., Blood 2016; Ramos et al., Blood 2012). HFE-related hereditary hemochromatosis is the most common type of hereditary iron overload disorder, with a high incidence of p.C282Y mutations in Northern European populations (Alexander and Kowdley, Genetics in Medicine, 2009). It is believed that impaired hepcidin production in the liver leads to iron overload in HFE-related hemochromatosis. In this case, iron overload often leads to complications such as cirrhosis and diabetes, as well as failure of other affected organs.

[0005] Consistent with its role in suppressing hepcidin expression, mutations in TMPRSS6 lead to conditions such as iron-refractory iron-deficiency anemia. In TMPRSS6- / - mice, hepcidin expression is significantly elevated, while in Hfe- / - mice, reduction of TMPRSS6 improves the iron overload phenotype (Du et al., Science 2008; Folgueras et al., Blood 2008; Finberg KE et al., Blood, 2011).

[0006] Such evidence suggests a role for targeting TMPRSS6 in treating diseases associated with iron overload. Current treatment options for such diseases are limited. Therefore, there is a need for methods of effectively treating conditions associated with iron overload, and the present invention meets this need. Summary of the invention

[0007] The present disclosure is based in part on the discovery of oligonucleotides (e.g., RNAi oligonucleotides) that reduce TMPRSS6 expression in vitro and in vivo. It has been shown that abnormal expression of TMPRSS6 is associated with changes in hepcidin expression and serum iron levels. As demonstrated herein, serum iron and serum iron saturation in vivo are reduced after administration of TMPRSS6 RNAi oligonucleotides. Without being bound by theory, inhibition of TMPRSS6 increases hepcidin expression, which subsequently reduces serum iron levels, thereby preventing iron overload. Therefore, without wishing to be bound by theory, oligonucleotides targeting TMPRSS6 can be used to treat diseases or conditions associated with hepcidin deficiency or inhibition.

[0008] Therefore, in some aspects, the present disclosure provides an RNAi oligonucleotide for reducing the expression of transmembrane serine protease 6 (TMPRSS6), the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises a complementary region that is complementary to the TMPRSS6 mRNA target sequence of any one of SEQ ID NOs: 661-852, and wherein the length of the complementary region is at least 15 consecutive nucleotides.

[0009] In some embodiments, the sense strand is 15 to 50 nucleotides in length. In some embodiments, the sense strand is 18 to 36 nucleotides in length.

[0010] In some embodiments, the antisense strand is 15 to 30 nucleotides in length.

[0011] In some embodiments, the antisense strand is 22 nucleotides in length, and the antisense strand and the sense strand form a duplex region of at least 19 nucleotides in length, optionally at least 20 nucleotides in length. In some embodiments, the complementary region is at least 19 contiguous nucleotides in length. In some embodiments, the complementary region is at least 20 contiguous nucleotides in length.

[0012] In some aspects, the present disclosure provides a double-stranded RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising:

[0013] (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is selected from the group consisting of SEQ ID NOs: 1-192, and

[0014] (ii) a sense strand of 19-50 nucleotides in length comprising a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0015] In some aspects, the present disclosure provides a double-stranded RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising:

[0016] (i) an antisense strand having a length of 19-30 nucleotides, wherein the antisense strand comprises a nucleotide sequence selected from SEQ ID NO: 1-192, and wherein the antisense strand is homologous to TMPRSS6

[0017] mRNA target sequence complementarity, and

[0018] (ii) a sense strand of 19-50 nucleotides in length comprising a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0019] In some embodiments, the 3' end of the sense strand comprises a stem-loop represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3-5 nucleotides in length between S1 and S2. In some embodiments, Lp is a tricyclic or tetracyclic loop. In some embodiments, Lp is a tetracyclic loop. In some embodiments, the tetracyclic loop comprises the sequence 5'-GAAA-3'. In some embodiments, the length of S1 and S2 is 1-10 nucleotides and has the same length. In some embodiments, the length of S1 and S2 is 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides. In some embodiments, the length of S1 and S2 is 6 nucleotides. In some embodiments, the stem-loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 856).

[0020] In some embodiments, the sense strand comprises a stem-loop represented as S1-Lp-S2 proximal to the 3' end of the sense strand, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3-5 nucleotides in length between S1 and S2. In some embodiments, Lp is a tricyclic or tetracyclic loop. In some embodiments, Lp is a tetracyclic loop. In some embodiments, the tetracyclic loop comprises the sequence 5'-GAAA-3'. In some embodiments, S1 and S2 are 1-10 nucleotides in length and have the same length. In some embodiments, S1 and S2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides in length. In some embodiments, S1 and S2 are 6 nucleotides in length. In some embodiments, the stem-loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 856).

[0021] In some embodiments, the antisense strand comprises a 3' overhang sequence of one or more nucleotides in length. In some embodiments, the overhang comprises a purine nucleotide. In some embodiments, the 3' overhang sequence is 2 nucleotides in length. In some embodiments, the 3' overhang is selected from AA, GG, AG and GA. In some embodiments, the overhang is GG or AA. In some embodiments, the overhang is GG.

[0022] In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2'-modification. In some embodiments, the 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid. In some embodiments, the modification is a 2'-modification selected from 2'-fluoro and 2'-O-methyl. In some embodiments, about 10-15%, 10%, 11%, 12%, 13%, 14% or 15% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise a 2'-fluoro modification. In some embodiments, the sense strand comprises 36 nucleotides from 5' to 3' positions 1-36, wherein positions 8-11 comprise a 2'-fluoro modification. In some embodiments, the antisense strand comprises 22 nucleotides from 5' to 3' positions 1-22, and wherein positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2'-fluoro modification.

[0023] In some embodiments, the oligonucleotide comprises at least one modified internucleotide bond. In some embodiments, the at least one modified internucleotide bond is a phosphorothioate bond. In some embodiments, the antisense strand (i) is between positions 1 and 2 and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, comprising a phosphorothioate bond, wherein positions are numbered 1-4 from 5' to 3'. In some embodiments, the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate bond between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5' to 3'. In some embodiments, the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate bond between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22. In some embodiments, the sense strand comprises a phosphorothioate bond between positions 1 and 2, wherein positions are numbered from 5' to 3'.

[0024] In some embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the phosphate analog is an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate, optionally wherein the phosphate analog is a 4'-phosphate analog comprising a 4'-oxymethylphosphonate.

[0025] In some embodiments, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol or a polypeptide. In some embodiments, the stem-loop comprises one or more targeting ligands conjugated to one or more nucleotides of the stem-loop. In some embodiments, the one or more targeting ligands are conjugated to one or more nucleotides of the ring. In some embodiments, the ring comprises 4 nucleotides numbered 1-4 from 5' to 3', wherein the nucleotides at positions 2, 3 and 4 each comprise one or more targeting ligands, wherein the targeting ligands are the same or different. In some embodiments, each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety. In some embodiments, up to 4 nucleotides of the Lp of the stem-loop are each conjugated to a monovalent GalNAc moiety.

[0026] In some embodiments, the complementary region is fully complementary to the mRNA target sequence. In some embodiments, the complementary region is partially complementary to the mRNA target sequence. In some embodiments, the complementary region contains no more than 4 mismatches with the mRNA target sequence.

[0027] In some embodiments, the complementary region is fully complementary to the TMPRSS6 mRNA target sequence at nucleotide positions 2-8 of the antisense strand, wherein the nucleotide positions are numbered from 5' to 3'. In some embodiments, the complementary region is fully complementary to the TMPRSS6 mRNA target sequence at nucleotide positions 2-11 of the antisense strand, wherein the nucleotide positions are numbered from 5' to 3'.

[0028] In some embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 579-580, 585-587, 590, and 595-597. In some embodiments, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 600-601, 606-608, 611, and 616-618.

[0029] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0030] a) SEQ ID NO: 579 and 600, respectively;

[0031] b) SEQ ID NO: 580 and 601, respectively;

[0032] c) SEQ ID NOs: 595 and 616, respectively;

[0033] d) SEQ ID NO: 590 and 611, respectively;

[0034] e) SEQ ID NOs: 596 and 617, respectively;

[0035] f) SEQ ID NOs: 597 and 618, respectively;

[0036] g) SEQ ID NOs: 585 and 606, respectively;

[0037] h) SEQ ID NOs: 586 and 607, respectively; and

[0038] i) are SEQ ID NOs: 587 and 608, respectively.

[0039] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0040] a) SEQ ID NO: 579 and 600, respectively;

[0041] b) SEQ ID NO: 580 and 601, respectively;

[0042] c) SEQ ID NOs: 590 and 611, respectively;

[0043] d) SEQ ID NOs: 597 and 618, respectively; and

[0044] e) are SEQ ID NOs: 586 and 607, respectively.

[0045] In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 579, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 600. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 580, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 601. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 590, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 611. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 597, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 618. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 586, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 607.

[0046] In some embodiments, the antisense strand is 22 nucleotides in length.

[0047] In some embodiments, the antisense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 600-601, 606-608, 611, and 616-618.

[0048] In some embodiments, the sense strand is 36 nucleotides in length.

[0049] In some embodiments, the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 579-580, 585-587, 590, and 595-597.

[0050] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0051] a) SEQ ID NO: 621 and 642, respectively;

[0052] b) SEQ ID NO: 622 and 643, respectively;

[0053] c) SEQ ID NOs: 637 and 658, respectively;

[0054] d) SEQ ID NOs: 632 and 653, respectively;

[0055] e) SEQ ID NOs: 638 and 659, respectively;

[0056] f) SEQ ID NOs: 639 and 660, respectively;

[0057] g) SEQ ID NOs: 627 and 648, respectively;

[0058] h) SEQ ID NOs: 628 and 649, respectively; and

[0059] i) are SEQ ID NOs: 629 and 650, respectively.

[0060] In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0061] a) SEQ ID NO: 621 and 642, respectively;

[0062] b) SEQ ID NO: 622 and 643, respectively;

[0063] c) SEQ ID NOs: 632 and 653, respectively;

[0064] d) SEQ ID NOs: 639 and 660, respectively; and

[0065] e) are SEQ ID NOs: 628 and 649, respectively.

[0066] In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 621, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 642. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 622, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 643. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 632, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 653. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 639, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 660. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 628, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 649.

[0067] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mGs][mG][mU][mG][mC][mU][mA][fC][fU][fC][fU][mG][mG][mU][mA][mU][mU][mU][mC][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3'(SEQ ID NO: 1) ID NO:621) and all modifications, and wherein the antisense strand comprises the sequence of 5'[MePhosphonate-4O-mUs][fGs][fAs][fA][fA][mU][fA][mC][mC][fA][mG][mA][mG][fU][mA][mG][mC][mA][mC][mCs][mGs][mG]-3' (SEQ ID NO:642) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein

[0068]

[0069] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5' [mGs][mC][mU][mA][mC][mU][mC][fU][fG][fG][fU][mA][mU][mU][mC][mC][mU][mA][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:622) and all modifications, and wherein the antisense strand comprises the sequence of 5'[MePhosphonate-4O-mUs][fUs][fAs][fG][fG][mA][fA][mA][mU][fA][mC][mC][mA][fG][mA][mG][mU][mA][mG][mCs][mGs][mG]-3' (SEQ ID NO:643) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein

[0070] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mCs][mU][mC][mA][mC][mC][mU][fG][fC][fU][fU][mC][mU][mC][mU][mG][mG][mU][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3'(SEQ ID NO: 1) ID NO:632) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fAs][fCs][fC][fA][mG][fA][mA][mG][fA][mA][mG][mC][fA][mG][mG][mU][mG][mA][mGs][mGs][mG]-3' (SEQ ID NO:653) and all modifications, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = thiophosphate, and wherein

[0071] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mAs][mG][mU][mG][mU][mG][mA][fA][fA][fG][fA][mC][mA][mU][mA][mG][mC][mU][mG][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]3'(SEQ ID NO: 1) ID NO:639) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fCs][fAs][fG][fC][mU][fA][mU][mG][fU][mC][mU][mU][fU][mC][mA][mC][mA][mC][mUs][mGs][mG]-3' (SEQ ID NO:660) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein

[0072] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mGs][mG][mG][mU][mG][mC][mA][fC][fU][fA][fU][mG][mG][mC][mU][mU][mG][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC] 3'(SEQ ID NO: 1) ID NO:628) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fUs][fAs][fC][fA][mA][fG][mC][mC][fA][mU][mA][mG][fU][mG][mC][mA][mC][mCs][mGs][mG]-3' (SEQ ID NO:649) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein

[0073] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 621 and the antisense strand comprises SEQ ID NO: 642, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a RNAi having Fig.14A The conjugate forms of the structures shown.

[0074] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 632 and the antisense strand comprises SEQ ID NO: 653, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a Fig. 14B The conjugate forms of the structures shown.

[0075] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 639 and the antisense strand comprises SEQ ID NO: 660, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a RNAi having Fig. 14C The conjugate forms of the structures shown.

[0076] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 628 and the antisense strand comprises SEQ ID NO: 649, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a RNAi having Fig.14D The conjugate forms of the structures shown.

[0077] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 621 and the antisense strand comprises SEQ ID NO: 642, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a RNAi having Fig. 10A -The form of the conjugate of the structure shown in B.

[0078] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 632 and the antisense strand comprises SEQ ID NO: 653, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a Fig.11A -The form of the conjugate of the structure shown in B.

[0079] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 639 and the antisense strand comprises SEQ ID NO: 660, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a RNAi having Fig. 12A -The form of the conjugate of the structure shown in B.

[0080] In some aspects, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 628 and the antisense strand comprises SEQ ID NO: 649, the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, wherein the RNAi is a RNAi having Fig.13A -The form of the conjugate of the structure shown in B.

[0081] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the RNAi oligonucleotide described herein and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0082] In some embodiments, the present disclosure provides a method for treating a subject having a disease, disorder, or condition associated with hepcidin deficiency or inhibition, the method comprising administering to the subject a therapeutically effective amount of an RNAi oligonucleotide or pharmaceutical composition described herein, thereby treating the subject.

[0083] In some embodiments, after administration of the RNAi oligonucleotide, hepcidin expression increases. In some embodiments, after administration of the RNAi oligonucleotide, iron saturation levels in serum decrease. In some embodiments, after administration of the RNAi oligonucleotide, serum iron levels decrease.

[0084] In some embodiments, the present disclosure provides a method of delivering an oligonucleotide to a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0085] In some embodiments, the present disclosure provides a method for reducing TMPRSS6 expression in a cell, a cell population, or a subject, the method comprising the steps of:

[0086] i. contacting the cell or cell population with an RNAi oligonucleotide as described herein or a pharmaceutical composition as described herein; or

[0087] ii. administering to the subject an RNAi oligonucleotide described herein or a pharmaceutical composition described herein.

[0088] In some embodiments, reducing TMPRSS6 expression includes reducing the amount or level of TMPRSS6 mRNA, the amount or level of membrane serine protease-2 (matriptase-2) protein, or both. In some embodiments, reducing TMPRSS6 expression results in increased hepcidin production. In some embodiments, reducing TMPRSS6 expression results in reduced serum iron levels. In some embodiments, reducing TMPRSS6 expression results in reduced serum iron saturation.

[0089] In some embodiments, the subject suffers from a disease, disorder, or condition associated with hepcidin deficiency or inhibition. In some embodiments, the disease, disorder, or condition associated with hepcidin deficiency is hemochromatosis or beta-thalassemia. In some embodiments, the disease, disorder, or condition associated with hepcidin inhibition is polycythemia vera.

[0090] In some embodiments, the RNAi oligonucleotide or pharmaceutical composition is administered in conjunction with a second composition or therapeutic agent.

[0091] In some embodiments, the present disclosure provides the use of the RNAi oligonucleotide or pharmaceutical composition described herein in the preparation of a medicament for treating a disease, disorder or condition associated with hepcidin deficiency or inhibition, optionally for treating hemochromatosis (e.g., hereditary hemochromatosis), polycythemia vera, or β-thalassemia.

[0092] In some embodiments, the present disclosure provides an RNAi oligonucleotide or pharmaceutical composition described herein, which is used or suitable for use in treating a disease, disorder or condition associated with hepcidin deficiency or inhibition, optionally for treating hemochromatosis (e.g., hereditary hemochromatosis), polycythemia vera, or β-thalassemia.

[0093] In some embodiments, the present disclosure provides a kit comprising an RNAi oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition.

[0094] In some embodiments, the present disclosure provides an RNAi oligonucleotide or pharmaceutical composition for use or suitable for use, or a kit described herein, wherein the disease, disorder or condition associated with hepcidin deficiency or inhibition is hemochromatosis (e.g., hereditary hemochromatosis), polycythemia vera, or β-thalassemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1A and Figure 1BThe graph provided depicts the percentage (%) of human TMPRSS6 mRNA remaining in the liver of mice exogenously expressing human TMPRSS6 (hydrodynamic injection model) after treatment with GalNAc-conjugated TMPRSS6 oligonucleotides. CD-1 mice were subcutaneously administered 1 mg / kg or 2 mg / kg of the indicated GalNAc-conjugated TMPRSS6 oligonucleotides formulated in PBS. Four days after administration, mice were hydrodynamically injected (HDI) with a DNA plasmid encoding human TMPRSS6. Using 3' qPCR assay ( Figure 1A ) and 5'qPCR assay ( Figure 1B ) Levels of human TMPRSS6 mRNA were determined from livers collected 18 hours later. Hs / Mf = construct is specific for human and monkey TMPRSS6; Hs-Mf-Mm = construct is specific for human, monkey and mouse TMPRSS6.

[0096] Figure 2 The figure provided depicts Figure 1A-1B The percentage (%) of mouse TMPRSS6 mRNA remaining in the liver of mice.

[0097] Figure 3A-3D Provided is a depiction of GalNAc-conjugated TMPRSS6 oligonucleotide-0416 ( Figure 3A )、-0651( Figure 3B )、-0831( Figure 3C ) and -1546( Figure 3D ) is a schematic diagram of the modification pattern of the nucleotide sequence of the nucleotide sequence of FIG. The sense strand includes a tetraloop structure consisting of nucleotides 27-30 in a strand consisting of 36 nucleotides. The antisense strand is complementary and includes an overhang consisting of 2 nucleotides.

[0098] Figure 4A-4F The graph provided depicts the percentage (%) of endogenous monkey TMPRSS6 mRNA remaining in the liver of NHPs (non-human primates) after treatment with GalNAc-conjugated TMPRSS6 oligonucleotides. Cynomolgus monkeys (Macaca fascicularis) were subcutaneously administered 1 mg / kg or 4 mg / kg of the indicated GalNAc-conjugated TMPRSS6 oligonucleotides formulated in PBS on days 0, 28, 56, 84, and 112. Figure 4A ), Day 28( Figure 4B )、Day 56( Figure 4C )、Day 84( Figure 4D ), Day 112( Figure 4E ) and Day 168 ( Figure 4F ) Liver samples were collected and levels of monkey TMPRSS6 mRNA were determined relative to PBS-treated animals using a qPCR assay.

[0099] Figure 5A-5F The graph provided depicts the percentage (%) of monkey endogenous hepcidin mRNA remaining in the liver of NHP (non-human primate) after treatment with GalNAc-conjugated TMPRSS6 oligonucleotides. Cynomolgus monkeys were subcutaneously administered 1 mg / kg or 4 mg / kg of the indicated GalNAc-conjugated TMPRSS6 oligonucleotides formulated in PBS on days 0, 28, 56, 84, and 112. On day -7 (1 week before administration) ( Figure 5A ), Day 28( Figure 5B )、Day 56( Figure 5C )、Day 84( Figure 5D ), Day 112( Figure 5E ) and Day 168 ( Fig. 5F ) Liver samples were collected and levels of monkey hepcidin mRNA were determined relative to PBS-treated animals using a qPCR assay.

[0100] Figure 6A-6F The provided graph depicts serum iron levels in monkeys after treatment with GalNAc-conjugated TMPRSS6 oligonucleotides. Cynomolgus monkeys were subcutaneously administered 1 mg / kg or 4 mg / kg of the indicated GalNAc-conjugated TMPRSS6 oligonucleotides formulated in PBS on days 0, 28, 56, 84, and 112. Fig. 6A ), Day 28( Figure 6B )、Day 56( Figure 6C )、Day 84( Fig.6D ), Day 112( Fig. 6E ) and Day 168 ( Fig. 6F ) Serum samples were collected and serum iron levels were determined.

[0101] Figure 7A-7B The graphs provided depict the difference in the expression of 1 mg / kg and 4 mg / kg, respectively, relative to PBS control treated animals. Figure 6A-6F Serum iron levels measured in .

[0102] Figure 8A-8F The provided graph depicts serum iron saturation levels in monkeys following treatment with GalNAc-conjugated TMPRSS6 oligonucleotides. Cynomolgus monkeys were subcutaneously administered 1 mg / kg or 4 mg / kg of the indicated GalNAc-conjugated TMPRSS6 oligonucleotides formulated in PBS on days 0, 28, 56, 84, and 112. Fig. 8A ), Day 28( Figure 8B )、Day 56( Figure 8C )、Day 84( Fig.8D ), Day 112( Fig. 8E ) and Day 168 ( Fig.8F ) Serum samples were collected and serum iron saturation was determined.

[0103] Figure 9A-9B The graphs provided depict the difference in the expression of 1 mg / kg and 4 mg / kg, respectively, relative to PBS control treated animals. Figure 8A-8F Serum iron saturation measured in .

[0104] Fig. 10A -B provides a chemical diagram of GalNAc-conjugated TMPRSS6 oligonucleotide-0416, where A and B represent Fig. 10A and Fig. 10B The key between.

[0105] Fig.11A -B provides a chemical diagram of GalNAc-conjugated TMPRSS6 oligonucleotide-0651, where A and B represent Fig.11A and Fig. 11B The key between.

[0106] Fig. 12A -B provides a chemical diagram of GalNAc-conjugated TMPRSS6 oligonucleotide-0831, where A and B represent Fig. 12A and Fig. 12B The key between.

[0107] Fig.13A -B provides a chemical diagram of GalNAc-conjugated TMPRSS6 oligonucleotide-1546, where A and B represent Fig.13A and Fig. 13B The key between.

[0108] Fig.14A -D provides GalNAc-conjugated TMPRSS6 oligonucleotide-0416 ( Fig.14A )、-0651( Fig. 14B )、-0831( Fig. 14C ) and -1546( Fig.14D )'s chemistry. DETAILED DESCRIPTION

[0109] Transmembrane serine protease 6 (TMPRSS6) encodes a type II transmembrane serine protease located on the cell surface, matriptase-2. Matriptase-2 protein plays a role in iron homeostasis by regulating hepcidin.

[0110] According to some aspects, the present disclosure provides oligonucleotides (e.g., RNAi oligonucleotides) that reduce TMPRSS6 expression in the liver. In some embodiments, the oligonucleotides provided herein are designed to treat diseases associated with hepcidin deficiency or inhibition. In some aspects, the present disclosure provides methods for treating diseases associated with hepcidin deficiency or inhibition by reducing TMPRSS6 expression in specific cells (e.g., liver cells) or organs (e.g., liver).

[0111] Oligonucleotide inhibitors targeting TMPRSS6 expression

[0112] TMPRSS6 target sequence

[0113] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) target a target sequence constituting TMPRSS6 mRNA. In some embodiments, the oligonucleotides described herein target a target sequence within a TMPRSS6 mRNA sequence. In some embodiments, the oligonucleotides described herein correspond to a target sequence within a TMPRSS6 mRNA sequence. In some embodiments, the oligonucleotides or portions, fragments, or strands thereof (e.g., the antisense strand or guide strand of a double-stranded (ds) RNAi oligonucleotide) bind to or anneal to a target sequence constituting TMPRSS6 mRNA, thereby inhibiting TMPRSS6 expression.

[0114] In some embodiments, the oligonucleotide targets a TMPRSS6 target sequence for the purpose of inhibiting TMPRSS6 expression in vivo. In some embodiments, the amount or degree to which an oligonucleotide targeting a TMPRSS6 target sequence inhibits TMPRSS6 expression is correlated with the efficacy of the oligonucleotide. In some embodiments, the amount or degree to which an oligonucleotide targeting a TMPRSS6 target sequence inhibits TMPRSS6 expression is correlated with the amount or degree of therapeutic benefit in a subject or patient treated with the oligonucleotide who suffers from a disease, disorder or condition associated with hepcidin deficiency or inhibition.

[0115] By examining the nucleotide sequences of mRNA encoding TMPRSS6, including the nucleotide sequences of mRNAs of multiple different species (e.g., humans, cynomolgus monkeys, and mice; see, e.g., Example 2), and as a result of in vitro and in vivo testing (see, e.g., Examples 2-5), it has been found that certain nucleotide sequences of TMPRSS6 mRNA are more susceptible to oligonucleotide-based inhibition than other sequences and can therefore be used as target sequences for oligonucleotides herein. In some embodiments, the TMPRSS6 target sequence comprises the sequence of any one of SEQ ID NOs: 661-852, or consists of the sequence of any one of SEQ ID NOs: 661-852. In some embodiments, the TMPRSS6 target sequence comprises the sequence set forth in SEQ ID NOs: 844, 841, 818, 794, or 762, or consists of the sequence set forth in SEQ ID NOs: 844, 841, 818, 794, or 762. In some embodiments, the TMPRSS6 target sequence comprises the sequence set forth in SEQ ID NO: 844, or consists of the sequence set forth in SEQ ID NO: 844. In some embodiments, the TMPRSS6 target sequence comprises or consists of the sequence set forth in SEQ ID NO: 841. In some embodiments, the TMPRSS6 target sequence comprises or consists of the sequence set forth in SEQ ID NO: 818. In some embodiments, the TMPRSS6 target sequence comprises or consists of the sequence set forth in SEQ ID NO: 794. In some embodiments, the TMPRSS6 target sequence comprises or consists of the sequence set forth in SEQ ID NO: 762.

[0116] TMPRSS6 targeting sequence

[0117] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have a complementary region complementary to TMPRSS6 mRNA (e.g., within the target sequence of TMPRSS6 mRNA) for the purpose of targeting TMPRSS6 mRNA in cells and inhibiting and / or reducing TMPRSS6 expression. In some embodiments, the oligonucleotides herein comprise a TMPRSS6 targeting sequence (e.g., the antisense strand or guide strand of an RNAi oligonucleotide) having a complementary region that binds or anneals to the TMPRSS6 target sequence by complementary (Watson-Crick) base pairing. The targeting sequence or complementary region generally has a suitable length and base content so that the oligonucleotide (or its strand) can bind or anneal to TMPRSS6 mRNA, thereby achieving the purpose of inhibiting and / or reducing TMPRSS6 expression. In some embodiments, the length of the targeting sequence or complementary region is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides. In some embodiments, the length of the targeting sequence or complementary region is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 18 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 19 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 20 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 21 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 22 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 23 nucleotides. In some embodiments, the length of the targeting sequence or complementary region is 24 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to a sequence of any one of SEQ ID NO:661-852, and the length of the targeting sequence or complementary region is 18 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to a sequence of any one of SEQ ID NO:661-852, and the length of the targeting sequence or complementary region is 19 nucleotides.In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NOs: 193-384, and the length of the targeting sequence or the complementary region is 20 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NOs: 193-384, and the length of the targeting sequence or the complementary region is 21 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NOs: 193-384, and the length of the targeting sequence or the complementary region is 22 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NOs: 193-384, and the length of the targeting sequence or the complementary region is 23 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region complementary to a sequence of any one of SEQ ID NOs: 193-384, and the length of the targeting sequence or the complementary region is 24 nucleotides.

[0118] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a targeting sequence or complementary region (e.g., the antisense strand or guide strand of a double-stranded oligonucleotide) that is fully complementary to a TMPRSS6 target sequence. In some embodiments, the targeting sequence or complementary region is partially complementary to a TMPRSS6 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is fully complementary to a TMPRSS6 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is partially complementary to a TMPRSS6 target sequence.

[0119] In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is fully complementary to the sequence of any one of SEQ ID NOs: 661-852. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is fully complementary to the sequence shown in SEQ ID NOs: 844, 841, 818, 794, and 762. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is partially complementary to the sequence of any one of SEQ ID NOs: 661-852. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is partially complementary to the sequence shown in SEQ ID NOs: 844, 841, 818, 794, and 762.

[0120] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence within a TMPRSS6 mRNA, wherein the contiguous nucleotide sequence is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20, or 18 to 19 nucleotides in length). In some embodiments, the oligonucleotides comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence within a TMPRSS6 mRNA, wherein the contiguous nucleotide sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence within a TMPRSS6 mRNA, wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence within TMPRSS6 mRNA, wherein the contiguous nucleotide sequence is 20 nucleotides in length.

[0121] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 661-852, optionally wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 844, 841, 818, 794, and 762, wherein the contiguous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotides comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 376, 373, 350, 326, and 294, wherein the contiguous nucleotide sequence is 20 nucleotides in length.

[0122] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a targeting sequence or complementary region that has one or more base pair (bp) mismatches with a corresponding TMPRSS6 target sequence. In some embodiments, the targeting sequence or complementary region may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with a corresponding TMPRSS6 target sequence, provided that the ability of the targeting sequence or complementary region to bind or anneal to TMPRSS6 mRNA under appropriate hybridization conditions and / or the ability of the oligonucleotide to inhibit TMPRSS6 expression is maintained. Alternatively, the targeting sequence or complementary region may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with a corresponding TMPRSS6 target sequence, provided that the ability of the targeting sequence or complementary region to bind or anneal to TMPRSS6 mRNA under appropriate hybridization conditions and / or the ability of the oligonucleotide to inhibit TMPRSS6 expression is maintained. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that has 1 mismatch with a corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 2 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 3 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 4 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with 5 mismatches with the corresponding target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region with more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) mismatches are continuously located (e.g., continuous 2, 3, 4, 5 or more mismatches), or wherein the mismatches are spread throughout the entire targeting sequence or complementary region. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that has more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) mismatches are located consecutively (e.g., consecutive 2, 3, 4, 5 or more mismatches), or wherein at least one or more non-mismatched base pairs are located between the mismatches, or a combination thereof. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a consecutive nucleotide sequence of any one of SEQ ID NOs: 661-852, wherein the targeting sequence or complementary region may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding TMPRSS6 target sequence.In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 661-852, wherein the targeting sequence or complementary region may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding TMPRSS6 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 661-852, wherein the targeting sequence or complementary region may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding TMPRSS6 target sequence. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 844, 841, 818, 794, and 762, wherein the targeting sequence or complementary region may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding TMPRSS6 target sequence.

[0123] Types of Oligonucleotides

[0124] A variety of oligonucleotide types and / or structures can be used to target TMPRSS6 in the methods herein, including but not limited to RNAi oligonucleotides, antisense oligonucleotides (ASOs), miRNAs, etc. Any oligonucleotide type described herein or elsewhere is contemplated for use as a framework to incorporate the TMPRSS6 targeting sequences herein for the purpose of inhibiting TMPRSS6 expression.

[0125] In some embodiments, the oligonucleotides herein inhibit TMPRSS6 expression by adding RNA interference (RNAi) pathways upstream or downstream of Dicer. For example, RNAi oligonucleotides have been developed, each of which has a size of about 19-25 nucleotides and has at least one 3' overhang consisting of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed, which are processed by Dicer to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work has produced extended dsRNAs, wherein at least one end of at least one chain extends beyond the duplex targeting region, including structures in which one chain comprises a thermodynamically stable tetracyclic structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO2010 / 033225). Such structures may include single-stranded (ss) stretches (on one or both sides of the molecule) as well as double-stranded (ds) stretches.

[0126] In some embodiments, the oligonucleotides herein are added to the RNAi pathway downstream of the participation of the Dicer (e.g., Dicer cleavage). In some embodiments, the oligonucleotides described herein are Dicer substrates. In some embodiments, after endogenous Dicer processing, a double-stranded nucleic acid of 19-23 nucleotides in length that can reduce TMPRSS6 expression is produced. In some embodiments, the oligonucleotide has an overhang (e.g., 1, 2, or 3 nucleotides in length) at the 3' end of the antisense strand. In some embodiments, the oligonucleotide (e.g., siRNA) comprises a guide strand of 21 nucleotides that is antisense to the target RNA and a complementary passenger strand, wherein the two strands anneal to form a 19-bp duplex and an overhang consisting of 2 nucleotides at either or both 3' ends. Longer oligonucleotide designs can also be used, including oligonucleotides with a 23 nucleotide guide strand and a 21 nucleotide passenger strand, with a blunt end on the right side of the molecule (3' end of the passenger strand / 5' end of the guide strand) and a 3'-guide strand overhang consisting of two nucleotides on the left side of the molecule (5' end of the passenger strand / 3' end of the guide strand). In such molecules, there is a 21 bp duplex region. See, e.g., U.S. Pat. Nos. 9,012,138, 9,012,621, and 9,193,753.

[0127] In some embodiments, the oligonucleotides herein include sense strands and antisense strands with a length of about 17 to 36 (e.g., 17 to 36, 20 to 25, or 21-23) nucleotides. In some embodiments, the oligonucleotides described herein include antisense strands with a length of 19-30 nucleotides and sense strands with a length of 19-50 nucleotides, wherein the antisense strand and the sense strand are separate strands, and they are formed at the 3' end of the antisense strand with an asymmetric duplex region of an overhang consisting of 1-4 nucleotides. In some embodiments, the oligonucleotides herein include sense strands and antisense strands with a length of about 19-22 nucleotides. In some embodiments, the sense strand and the antisense strand are equal in length. In some embodiments, the oligonucleotides include sense strands and antisense strands, so that there are 3'-overhangs on the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, for oligonucleotides having sense and antisense strands each ranging in length from about 21-23 nucleotides, the 3' overhang on the sense strand, antisense strand, or both is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has a guide strand of 22 nucleotides and a passenger strand of 20 nucleotides with a blunt end on the right side of the molecule (3' end of passenger strand / 5' end of guide strand) and a 3'-guide strand overhang consisting of 2 nucleotides on the left side of the molecule (5' end of passenger strand / 3' end of guide strand). In such molecules, there is a 20 bp duplex region.

[0128] Other oligonucleotide designs for use with the compositions and methods herein include: 16-mer siRNA (see, e.g., Nucleic Acids in Chemistry and Biology, Blackburn (ed.), ROYAL SOCIETY OF CHEMISTRY, 2006), shRNA (e.g., having a stem of 19 bp or less; see, e.g., Moore et al. (2010) Methods Mol. Biol. 629:141-158), blunt-ended siRNA (e.g., 19 bp in length; see, e.g., Kraynack & Baker (2006) RNA 12:163-176), asymmetric siRNA (aiRNA; see, e.g., Sun et al. (2008) Nat. Biotechnol. 26:1379-82), asymmetric shorter duplex siRNA (see, e.g., Chang et al. (2009) Mol. Ther. 17:725-32), fork siRNA (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-98), single-stranded siRNA (Elsner (2012) Nat. Biotechnol. 30:1063), dumbbell-shaped circular siRNA (see, e.g., Abe et al. (2007) J. Am. Chem. Soc. 129:15108-09), and small internal segmented interfering RNA (siRNA; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35:5886-97). Other non-limiting examples of oligonucleotide structures that can be used in some embodiments to reduce or inhibit TMPRSS6 expression are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al. (2002) EMBO J. 21:4671-79; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0129] In addition, in some embodiments, the oligonucleotides used herein to reduce or inhibit TMPRSS6 expression are single-stranded (ss). Such structures may include, but are not limited to, single-stranded RNAi molecules. Recent efforts have demonstrated the activity of single-stranded RNAi molecules (see, e.g., Matsui et al. (2016) Mol. Ther. 24: 946-55). However, in some embodiments, the oligonucleotides herein are antisense oligonucleotides (ASOs). Antisense oligonucleotides are single-stranded oligonucleotides having a nucleobase sequence, which, when written in the 5' to 3' direction, comprises the reverse complementary sequence of the targeted segment of a specific nucleic acid and is appropriately modified (e.g., as a gapmer) to induce RNase H-mediated cleavage of its target RNA in a cell, or (e.g., as a mixer) to inhibit translation of a target mRNA in a cell. ASOs for use herein can be modified in any suitable manner known in the art, including, for example, as shown in U.S. Pat. No. 9,567,587 (including, for example, length, sugar moieties of nucleobases (pyrimidines, purines), and changes in heterocyclic moieties of nucleobases). In addition, ASOs have been used to reduce the expression of specific target genes for decades (see, for example, Bennett et al. (2017) Annu. Rev. Pharmacol. 57: 81-105).

[0130] In some embodiments, the antisense oligonucleotide shares a complementary region with TMPRSS6 mRNA. In some embodiments, the antisense oligonucleotide targets various regions of the human TMPRSS6 gene identified as NM_001289000.2. In some embodiments, the antisense oligonucleotide has a length of 15-50 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 15-25 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 22 nucleotides. In some embodiments, the antisense oligonucleotide is complementary to any one of SEQ ID NO: 661-852. In some embodiments, the antisense oligonucleotide has a length of at least 15 consecutive nucleotides. In some embodiments, the antisense oligonucleotide has a length of at least 19 consecutive nucleotides. In some embodiments, the antisense oligonucleotide has a length of at least 20 consecutive nucleotides. In some embodiments, the antisense oligonucleotide is different from the target sequence by 1, 2 or 3 nucleotides.

[0131] Double-stranded oligonucleotide

[0132] In some aspects, the present disclosure provides double-stranded (ds) RNAi oligonucleotides for targeting TMPRSS6 mRNA and inhibiting TMPRSS6 expression (e.g., via an RNAi pathway), comprising a sense strand (also referred to herein as a passenger strand) and an antisense strand (also referred to herein as a guide strand). In some embodiments, the sense strand and the antisense strand are separate strands and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked. In some embodiments, the sense strand and the antisense strand form a duplex region, wherein the sense strand and the antisense strand, or portions thereof, bind to each other in a complementary manner (e.g., by Watson-Crick base pairing).

[0133] In some embodiments, the first region (R1) of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, the length of D1 is at least about 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides. In some embodiments, the length of D1 is in the range of about 12 to 30 nucleotides (e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30, or 21 to 30 nucleotides in length). In some embodiments, the length of D1 is at least 12 nucleotides (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In some embodiments, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, D1 is 20 nucleotides in length. In some embodiments, D1 comprising a sense strand and an antisense strand does not span the entire length of the sense strand and / or antisense strand. In some embodiments, D1 comprising a sense strand and an antisense strand spans the entire length of the sense strand or the antisense strand or both. In certain embodiments, D1 comprising a sense strand and an antisense strand spans the entire length of both the sense strand and the antisense strand.

[0134] In some embodiments, the sense strand has a second region (R2), wherein R2 comprises a first subregion (S1), a loop (Lp) such as a tetraloop (tetraLp) or a triloop (triLp), and a second subregion (S2), wherein Lp is located between S1 and S2, and wherein S1 and S2 form a second duplex (D2). D2 may have different lengths. In some embodiments, the length of D2 is about 1-6 bp. In some embodiments, the length of D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, or 4-5 bp. In some embodiments, the length of D2 is 1, 2, 3, 4, 5, or 6 bp. In some embodiments, the length of D2 is 6 bp.

[0135] In some embodiments, the oligonucleotides provided herein comprise a sense strand and an antisense strand, the sense strand comprising the sequence of any one of SEQ ID NOs: 193-384, and the antisense strand comprising the sequence of any one of SEQ ID NOs: 385-576. In some embodiments, the oligonucleotides provided herein comprise a sense strand and an antisense strand, the sense strand comprising the sequence of any one of SEQ ID NOs: 661-852, and the antisense strand comprising the sequence of any one of SEQ ID NOs: 1-192.

[0136] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand comprising the sequence of any one of SEQ ID NOs: 577-597 and an antisense strand comprising the sequence of any one of SEQ ID NOs: 598-618, as arranged in Table 4.

[0137] In some embodiments, an oligonucleotide provided herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0138] a) SEQ ID NO: 579 and 600, respectively;

[0139] b) SEQ ID NO: 580 and 601, respectively;

[0140] c) SEQ ID NOs: 595 and 616, respectively;

[0141] d) SEQ ID NO: 590 and 611, respectively;

[0142] e) SEQ ID NOs: 596 and 617, respectively;

[0143] f) SEQ ID NOs: 597 and 618, respectively;

[0144] g) SEQ ID NOs: 585 and 606, respectively;

[0145] h) SEQ ID NOs: 586 and 607, respectively; and

[0146] i) are SEQ ID NOs: 587 and 608, respectively.

[0147] In some embodiments, an oligonucleotide provided herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0148] a) SEQ ID NO: 579 and 600, respectively;

[0149] b) SEQ ID NO: 580 and 601, respectively;

[0150] c) SEQ ID NOs: 590 and 611, respectively;

[0151] d) SEQ ID NOs: 597 and 618, respectively; and

[0152] e) are SEQ ID NOs: 586 and 607, respectively.

[0153] In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 579 and the antisense strand comprises the sequence of SEQ ID NO: 600. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 580 and the antisense strand comprises the sequence of SEQ ID NO: 601. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 590 and the antisense strand comprises the sequence of SEQ ID NO: 611. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 597 and the antisense strand comprises the sequence of SEQ ID NO: 618. In some embodiments, the sense strand comprises the sequence of SEQ ID NO: 586 and the antisense strand comprises the sequence of SEQ ID NO: 607.

[0154] It should be understood that in some embodiments, reference may be made to the sequences presented in the sequence listing when describing the structure of an oligonucleotide (e.g., an RNAi oligonucleotide) or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more substituted nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications compared to the specified sequence, while still retaining complementary properties that are substantially identical or similar to the specified sequence.

[0155] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, which, when acted upon by a cutting enzyme, produces an antisense strand that is incorporated into a mature RISC. In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, which, when acted upon by a cutting enzyme, produces an antisense strand that is incorporated into a mature RISC. In some embodiments, the sense strand of the 25 nucleotides comprises a sequence selected from SEQ ID NO: 193-384. In some embodiments, the antisense strand of the 27 nucleotides comprises a sequence selected from SEQ ID NO: 385-576. In some embodiments, the sense strand of the oligonucleotide is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand of the oligonucleotide is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides). In some embodiments, the sense strand of the oligonucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 577-597, wherein the nucleotide sequence is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand of the oligonucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 577-597, wherein the nucleotide sequence is longer than 25 nucleotides (eg, 26, 27, 28, 29, or 30 nucleotides).

[0156] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have a 5' end that is less thermodynamically stable than the other 5' end. In some embodiments, asymmetric oligonucleotides are provided that include a flat end at the 3' end of the sense strand and a 3'-overhang at the 3' end of the antisense strand. In some embodiments, the 3'-overhang on the antisense strand is about 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). In some embodiments, the oligonucleotide has an overhang comprising two (2) nucleotides at the 3' end of the antisense (guide) strand. However, other overhangs are also possible. In some embodiments, the overhang is a 3'-overhang comprising 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or a length of 1, 2, 3, 4, 5 or 6 nucleotides. However, in some embodiments, the overhang is a 5'-overhang comprising 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or a length of 1, 2, 3, 4, 5 or 6 nucleotides. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 661-852, and a 5'-overhang of 1 to 6 nucleotides in length. In some embodiments, the oligonucleotide comprises a sense strand, the sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 577-597, wherein the oligonucleotide comprises a 5'-overhang of 1 to 6 nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense strand, the antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 598-618, wherein the oligonucleotide comprises a 5'-overhang of 1 to 6 nucleotides in length. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 577-597, the antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 598-618, wherein the oligonucleotide comprises a 5'-overhang of 1 to 6 nucleotides in length.

[0157] In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand are modified. In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand are complementary to the target mRNA (e.g., TMPRSS6 mRNA). In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide herein are unpaired. In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide herein comprise unpaired guanine (e.g., 5'-GG-3'). In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide herein are not complementary to the target mRNA. In some embodiments, the two (2) terminal nucleotides on each 3' end of the oligonucleotide are guanine (GG). In some embodiments, one or both of the two (2) terminal GG nucleotides on each 3' end of the oligonucleotide herein are not complementary to the target mRNA. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 661-852, wherein the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide herein comprise unpaired GG. In some embodiments, the oligonucleotide comprises an antisense strand, the antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1-192, wherein the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide comprise unpaired GG. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence selected from SEQ ID NOs: 577-597, the antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 598-618, wherein the two (2) terminal nucleotides on the 3' end of the antisense strand of the oligonucleotide comprise unpaired GG.

[0158] In some embodiments, there are one or more (e.g., 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand that constitute the oligonucleotide herein (e.g., RNAi oligonucleotide). If there are more than one mismatches between the sense strand and the antisense strand, they may be positioned consecutively (e.g., 2, 3, or more consecutively), or interspersed throughout the complementary region. In some embodiments, the 3' end of the sense strand comprises one or more mismatches. In some embodiments, two (2) mismatches are incorporated at the 3' end of the sense strand. In some embodiments, base mismatches or segment destabilization at the 3' end of the sense strand of the oligonucleotide herein improve or increase the effectiveness of the oligonucleotide. In some embodiments, the sense strand and antisense strand of the oligonucleotide herein comprise a nucleotide sequence selected from the group consisting of:

[0159] a) SEQ ID NO: 579 and 600, respectively;

[0160] b) SEQ ID NO: 580 and 601, respectively;

[0161] c) SEQ ID NOs: 595 and 616, respectively;

[0162] d) SEQ ID NO: 590 and 611, respectively;

[0163] e) SEQ ID NOs: 596 and 617, respectively;

[0164] f) SEQ ID NOs: 597 and 618, respectively;

[0165] g) SEQ ID NOs: 585 and 606, respectively;

[0166] h) SEQ ID NOs: 586 and 607, respectively; and

[0167] i) SEQ ID NO: 587 and 608, respectively,

[0168] wherein there are one or more (eg, 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand.

[0169] In some embodiments, an oligonucleotide provided herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0170] a) SEQ ID NO: 579 and 600, respectively;

[0171] b) SEQ ID NO: 580 and 601, respectively;

[0172] c) SEQ ID NOs: 590 and 611, respectively;

[0173] d) SEQ ID NOs: 597 and 618, respectively; and

[0174] e) SEQ ID NO: 586 and 607, respectively,

[0175] wherein there are one or more (eg, 1, 2, 3, 4, or 5) mismatches between the sense strand and the antisense strand.

[0176] Antisense strand

[0177] In some embodiments, the antisense strand of an oligonucleotide (e.g., RNAi oligonucleotide) herein is referred to as a "guide strand". For example, the antisense strand engages with an RNA-induced silencing complex (RISC) and binds to an Argonaute protein such as Ago2, or engages or binds to one or more similar factors, and guides the silencing of a target gene, and the antisense strand is referred to as a guide strand. In some embodiments, a sense strand comprising a complementary region complementary to a guide strand is referred to herein as a "passenger strand".

[0178] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise an antisense strand of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotides comprise an antisense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, the oligonucleotide comprises an antisense strand ranging from about 12 to about 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides in length. In some embodiments, the antisense strand of any of the oligonucleotides disclosed herein is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense strand of 22 nucleotides in length.

[0179] In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 (e.g., RNAi oligonucleotides) comprise an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 385-576 or consisting of a sequence as set forth in any one of SEQ ID NOs: 385-576. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 385-576. In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 comprise an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 598-618 or consisting of a sequence as set forth in any one of SEQ ID NOs: 598-618. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 598-618. In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 comprise an antisense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 598-618. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 600, 601, 611, 618, and 607. In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 comprise an antisense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 600, 601, 611, 618, and 607.

[0180] In some embodiments, the oligonucleotides herein comprise an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 1-192. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising a nucleotide sequence selected from SEQ ID NOs: 184, 181, 158, 134, and 102.

[0181] Sense strand

[0182] In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 mRNA and inhibiting TMPRSS6 expression (e.g., RNAi oligonucleotides) comprise a sense strand sequence as shown in any one of SEQ ID NOs: 661-852. In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 mRNA and inhibiting TMPRSS6 expression (e.g., RNAi oligonucleotides) comprise a sense strand sequence as shown in any one of SEQ ID NOs: 193-384. In some embodiments, the oligonucleotides herein have a sense strand consisting of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as shown in any one of SEQ ID NOs: 193-384. In some embodiments, the oligonucleotides herein have a sense strand consisting of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 661-852. In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 mRNA and inhibiting TMPRSS6 expression comprise a sense strand sequence as set forth in any one of SEQ ID NOs: 577-597. In some embodiments, the oligonucleotides herein have a sense strand consisting of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs: 577-597. In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 mRNA and inhibiting TMPRSS6 expression comprise a sense strand sequence as shown in any one of SEQ ID NOs: 579, 580, 590, 597, and 586. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as shown in any one of SEQ ID NOs: 579, 580, 590, 597, and 586. In some embodiments, the oligonucleotides disclosed herein for targeting TMPRSS6 mRNA and inhibiting TMPRSS6 expression comprise a sense strand sequence as shown in any one of SEQ ID NOs: 844, 841, 818, 794, and 762.In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) consecutive nucleotides of a sequence as shown in any one of SEQ ID NOs: 844, 841, 818, 794, and 762.

[0183] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand (or passenger strand) of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotides herein comprise a sense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the oligonucleotides herein comprise a sense strand ranging in length from about 12 to about 50 (e.g., 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand of 15 to 50 nucleotides in length. In some embodiments, the oligonucleotides herein comprise a sense strand of 18 to 36 nucleotides in length. In some embodiments, the oligonucleotides herein comprise a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotides herein comprise a sense strand of 36 nucleotides in length.

[0184] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) include a sense strand, which includes a stem-loop structure at the proximal side of the 3' end of the sense strand. In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) include a sense strand, which includes a stem-loop structure at the 3' end of the first region (R1) of the sense strand. In some embodiments, the stem-loop is formed by intrachain base pairing. In some embodiments, the sense strand includes a stem-loop structure at the proximal side of its 5' end. In some embodiments, the sense strand includes a stem-loop structure at the 5' end of the first region (R1). In some embodiments, the stem of the stem-loop includes a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length. In some embodiments, the stem of the stem-loop includes a duplex of 2 nucleotides in length. In some embodiments, the stem of the stem-loop includes a duplex of 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 8 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 9 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 10 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 12 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 14 nucleotides in length.

[0185] In some embodiments, the stem-loop provides protection of the oligonucleotide from degradation (e.g., enzymatic degradation), promotes or improves targeting and / or delivery to a target cell, tissue, or organ (e.g., liver), or both. For example, in some embodiments, the loop of the stem-loop is composed of nucleotides comprising one or more modifications that promote, improve, or increase targeting to a target mRNA (e.g., TMPRSS6 mRNA), inhibition of target gene expression (e.g., TMPRSS6 expression), and / or delivery, uptake, and / or penetration into a target cell, tissue, or organ (e.g., liver), or a combination thereof. In some embodiments, the stem-loop itself or modifications to the stem-loop do not affect or substantially affect the inherent gene expression inhibition activity of the oligonucleotide, but promote, improve, or increase the stability of the oligonucleotide (e.g., providing protection against degradation) and / or its delivery, uptake, and / or penetration into a target cell, tissue, or organ (e.g., liver). In certain embodiments, the oligonucleotide herein comprises a sense strand comprising (e.g., at its 3' end, proximal to its 3' end, and / or at the 3' end of the first region (R1) of the sense strand) a stem-loop as follows: S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a single-stranded loop of linked nucleotides of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length) between S1 and S2. In some embodiments, the length of the loop (Lp) is 3 nucleotides (referred to herein as a "triloop"). In some embodiments, the length of the loop (Lp) is 4 nucleotides (referred to herein as a "tetraloop"). In some embodiments, the length of the loop (Lp) is 5 nucleotides. In some embodiments, the length of the loop (Lp) is 6 nucleotides. In some embodiments, the length of the loop (Lp) is 7 nucleotides. In some embodiments, the length of the loop (Lp) is 8 nucleotides. In some embodiments, the length of the loop (Lp) is 9 nucleotides. In some embodiments, the loop (Lp) is 10 nucleotides in length.

[0186] In some embodiments, the tetraloop comprises the sequence 5'-GAAA-3'.In some embodiments, the stem-loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 856).

[0187] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 661-852, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end, proximal to its 3' end, and / or at the 3' end of the first region (R1) of the sense strand) a stem-loop as follows: S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a single-stranded loop of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length) between S1 and S2. In some embodiments, the oligonucleotide comprises a targeting sequence or a complementary region that is complementary to a contiguous nucleotide sequence of any one of SEQ ID NOs: 661-852, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end, proximal to its 3' end, and / or at the 3' end of the first region (R1) of the sense strand) a stem-loop as follows: S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a single-stranded loop of 4 nucleotides in length between S1 and S2.

[0188] In some embodiments, the ring (Lp) of the stem-loop with structure S1-Lp-S2 as described herein is a tri-loop (triLp). In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region complementary to a continuous nucleotide sequence of any one of SEQ ID NO: 661-852, and a tri-loop. In some embodiments, the tri-loop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands) and combinations thereof.

[0189] In some embodiments, the ring (Lp) of the stem-ring with structure S1-Lp-S2 as described above is a tetraloop (tetraLp), as described in U.S. Pat. No. 10,131,912, which is incorporated herein by reference. In some embodiments, the oligonucleotides herein comprise a targeting sequence or complementary region complementary to a continuous nucleotide sequence of any one of SEQ ID NO: 661-852, and a tetraloop. In some embodiments, the tetraloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands) and combinations thereof.

[0190] Duplex length

[0191] In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is in the range of 12-30 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 12 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 13 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 14 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 15 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 16 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 17 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 18 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 19 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 20 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 21 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 22 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 23 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 24 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 25 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 26 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 27 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 28 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 29 nucleotides. In some embodiments, the length of the duplex formed between the sense strand and the antisense strand is 30 nucleotides.In some embodiments, the duplex formed between the sense strand and the antisense strand does not span the entire length of the sense strand and / or the antisense strand. In some embodiments, the duplex between the sense strand and the antisense strand spans the entire length of the sense strand or the antisense strand. In some embodiments, the duplex between the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:.

[0192] a) SEQ ID NO: 579 and 600, respectively;

[0193] b) SEQ ID NO: 580 and 601, respectively;

[0194] c) SEQ ID NOs: 595 and 616, respectively;

[0195] d) SEQ ID NO: 590 and 611, respectively;

[0196] e) SEQ ID NOs: 596 and 617, respectively;

[0197] f) SEQ ID NOs: 597 and 618, respectively;

[0198] g) SEQ ID NOs: 585 and 606, respectively;

[0199] h) SEQ ID NOs: 586 and 607, respectively; and

[0200] i) SEQ ID NO: 587 and 608, respectively,

[0201] wherein the length of the duplex formed between the sense strand and the antisense strand is in the range of 12-30 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0202] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0203] a) SEQ ID NO: 579 and 600, respectively;

[0204] b) SEQ ID NO: 580 and 601, respectively;

[0205] c) SEQ ID NOs: 590 and 611, respectively;

[0206] d) SEQ ID NOs: 597 and 618, respectively; and

[0207] e) SEQ ID NO: 586 and 607, respectively,

[0208] wherein the length of the duplex formed between the sense strand and the antisense strand is in the range of 12-30 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length).

[0209] Oligonucleotide ends

[0210] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) include a sense strand and an antisense strand, wherein the end of any one or both strands includes a flat end. In some embodiments, the oligonucleotides herein include a sense strand and an antisense strand, and the sense strand and the antisense strand are separate strands that form an asymmetric duplex region with an overhang at the 3' end of the antisense strand. In some embodiments, the oligonucleotides herein include a sense strand and an antisense strand, wherein the end of any one or both strands includes an overhang containing one or more nucleotides. In some embodiments, the one or more nucleotides constituting the overhang are unpaired nucleotides. In some embodiments, the oligonucleotides herein include a sense strand and an antisense strand, wherein the 3' end of the sense strand and the 5' end of the antisense strand include a flat end. In some embodiments, the oligonucleotides herein include a sense strand and an antisense strand, wherein the 5' end of the sense strand and the 3' end of the antisense strand include a flat end.

[0211] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 3' end of any one or both strands comprises a 3'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the sense strand comprises a 3'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 3'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise a 3'-overhang containing one or more nucleotides.

[0212] In some embodiments, the 3'-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 3' overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides in length. In some embodiments, the 3'-overhang is one (1) nucleotide in length. In some embodiments, the 3'-overhang is two (2) nucleotides in length. In some embodiments, the 3'-overhang is three (3) nucleotides in length. In some embodiments, the 3'-overhang is four (4) nucleotides in length. In some embodiments, the 3'-overhang is five (5) nucleotides in length. In some embodiments, the 3'-overhang is six (6) nucleotides in length. In some embodiments, the 3'-overhang is seven (7) nucleotides in length. In some embodiments, the 3'-overhang is eight (8) nucleotides in length. In some embodiments, the 3'-overhang is nine (9) nucleotides in length. In some embodiments, the 3'-overhang is ten (10) nucleotides in length. In some embodiments, the 3'-overhang is eleven (11) nucleotides in length. In some embodiments, the 3'-overhang is twelve (12) nucleotides in length. In some embodiments, the 3'-overhang is thirteen (13) nucleotides in length. In some embodiments, the 3'-overhang is fourteen (14) nucleotides in length. In some embodiments, the 3'-overhang is fifteen (15) nucleotides in length. In some embodiments, the 3'-overhang is sixteen (16) nucleotides in length. In some embodiments, the 3'-overhang is seventeen (17) nucleotides in length. In some embodiments, the 3'-overhang is eighteen (18) nucleotides in length. In some embodiments, the 3'-overhang is nineteen (19) nucleotides in length. In some embodiments, the 3'-overhang is twenty (20) nucleotides in length.

[0213] In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0214] a) SEQ ID NO: 579 and 600, respectively;

[0215] b) SEQ ID NO: 580 and 601, respectively;

[0216] c) SEQ ID NOs: 595 and 616, respectively;

[0217] d) SEQ ID NO: 590 and 611, respectively;

[0218] e) SEQ ID NOs: 596 and 617, respectively;

[0219] f) SEQ ID NOs: 597 and 618, respectively;

[0220] g) SEQ ID NOs: 585 and 606, respectively;

[0221] h) SEQ ID NOs: 586 and 607, respectively; and

[0222] i) SEQ ID NO: 587 and 608, respectively,

[0223] and wherein the antisense strand comprises a 3'-overhang of about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides in length), optionally wherein the 3'-overhang is two (2) nucleotides in length.

[0224] In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0225] a) SEQ ID NO: 579 and 600, respectively;

[0226] b) SEQ ID NO: 580 and 601, respectively;

[0227] c) SEQ ID NOs: 590 and 611, respectively;

[0228] d) SEQ ID NOs: 597 and 618, respectively; and

[0229] e) SEQ ID NO: 586 and 607, respectively,

[0230] and wherein the antisense strand comprises a 3'-overhang of about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides in length), optionally wherein the 3'-overhang is two (2) nucleotides in length.

[0231] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 5' end of any one or both strands comprises a 5'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the sense strand comprises a 5'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-overhang containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise a 5'-overhang containing one or more nucleotides.

[0232] In some embodiments, the 5'-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 5' overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides in length. In some embodiments, the 5'-overhang is one (1) nucleotide in length. In some embodiments, the 5'-overhang is two (2) nucleotides in length. In some embodiments, the 5'-overhang is three (3) nucleotides in length. In some embodiments, the 5'-overhang is four (4) nucleotides in length. In some embodiments, the 5'-overhang is five (5) nucleotides in length. In some embodiments, the 5'-overhang is six (6) nucleotides in length. In some embodiments, the 5'-overhang is seven (7) nucleotides in length. In some embodiments, the 5'-overhang is eight (8) nucleotides in length. In some embodiments, the 5'-overhang is nine (9) nucleotides in length. In some embodiments, the 5'-overhang is ten (10) nucleotides in length. In some embodiments, the 5'-overhang is eleven (11) nucleotides in length. In some embodiments, the 5'-overhang is twelve (12) nucleotides in length. In some embodiments, the 5'-overhang is thirteen (13) nucleotides in length. In some embodiments, the 5'-overhang is fourteen (14) nucleotides in length. In some embodiments, the 5'-overhang is fifteen (15) nucleotides in length. In some embodiments, the 5'-overhang is sixteen (16) nucleotides in length. In some embodiments, the 5'-overhang is seventeen (17) nucleotides in length. In some embodiments, the 5'-overhang is eighteen (18) nucleotides in length. In some embodiments, the 5'-overhang is nineteen (19) nucleotides in length. In some embodiments, the 5'-overhang is twenty (20) nucleotides in length.

[0233] In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0234] a) SEQ ID NO: 579 and 600, respectively;

[0235] b) SEQ ID NO: 580 and 601, respectively;

[0236] c) SEQ ID NOs: 595 and 616, respectively;

[0237] d) SEQ ID NO: 590 and 611, respectively;

[0238] e) SEQ ID NOs: 596 and 617, respectively;

[0239] f) SEQ ID NOs: 597 and 618, respectively;

[0240] g) SEQ ID NOs: 585 and 606, respectively;

[0241] h) SEQ ID NOs: 586 and 607, respectively; and

[0242] i) SEQ ID NO: 587 and 608, respectively,

[0243] and wherein the antisense strand comprises a 5'-overhang of about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides in length), optionally wherein the 5'-overhang is two (2) nucleotides in length.

[0244] In some embodiments, an oligonucleotide disclosed herein (e.g., an RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0245] a) SEQ ID NO: 579 and 600, respectively;

[0246] b) SEQ ID NO: 580 and 601, respectively;

[0247] c) SEQ ID NOs: 590 and 611, respectively;

[0248] d) SEQ ID NOs: 597 and 618, respectively; and

[0249] e) SEQ ID NO: 586 and 607, respectively,

[0250] and wherein the antisense strand comprises a 5'-overhang of about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides in length), optionally wherein the 5'-overhang is two (2) nucleotides in length.

[0251] In some embodiments, one or more (e.g., 2, 3, 4, 5 or more) nucleotides constituting the 3' end or 5' end of the sense strand and / or antisense strand are modified. For example, in some embodiments, one or both terminal nucleotides at the 3' end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3' end of the antisense strand is modified so that it comprises a 2' modification, or it comprises a 2'-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3' end of the antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3' end of the antisense strand are not complementary to the target.

[0252] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the 3' end of the sense strand comprises a stem-loop as described herein, and the 3' end of the antisense strand comprises a 3'-overhang as described herein. In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand forming a gapped tetraloop structure as described herein, wherein the 3' end of the sense strand comprises a stem-loop, wherein the loop is a tetraloop as described herein, and wherein the 3' end of the antisense strand comprises a 3'-overhang as described herein. In some embodiments, the 3'-overhang is two (2) nucleotides in length. In some embodiments, both (2) nucleotides constituting the 3'-overhang comprise a guanine (G) nucleobase. One or both of the nucleotides constituting the 3'-overhang of the antisense strand may not be complementary to the target mRNA. Typically, one or both of the nucleotides constituting the 3'-overhang of the antisense strand are not complementary to the target mRNA.

[0253] Oligonucleotide modification

[0254] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise modifications. Oligonucleotides (e.g., RNAi oligonucleotides) can be modified in a variety of ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other features associated with treatment or research use.

[0255] In some embodiments, the modification is a modified sugar. In some embodiments, the modification is a 5'-terminal phosphate group. In some embodiments, the modification is a modified internucleotide linkage. In some embodiments, the modification is a modified base. In some embodiments, the oligonucleotides described herein may comprise any one of the modifications described herein or any combination thereof. For example, in some embodiments, the oligonucleotides described herein comprise at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base. In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0256] a) SEQ ID NO: 579 and 600, respectively;

[0257] b) SEQ ID NO: 580 and 601, respectively;

[0258] c) SEQ ID NOs: 595 and 616, respectively;

[0259] d) SEQ ID NO: 590 and 611, respectively;

[0260] e) SEQ ID NOs: 596 and 617, respectively;

[0261] f) SEQ ID NOs: 597 and 618, respectively;

[0262] g) SEQ ID NOs: 585 and 606, respectively;

[0263] h) SEQ ID NOs: 586 and 607, respectively; and

[0264] i) SEQ ID NO: 587 and 608, respectively,

[0265] wherein the oligonucleotide comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide linkage and at least one modified base.

[0266] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0267] a) SEQ ID NO: 579 and 600, respectively;

[0268] b) SEQ ID NO: 580 and 601, respectively;

[0269] c) SEQ ID NOs: 590 and 611, respectively;

[0270] d) SEQ ID NOs: 597 and 618, respectively; and

[0271] e) SEQ ID NO: 586 and 607, respectively,

[0272] wherein the oligonucleotide comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide linkage and at least one modified base.

[0273] The number of modifications on an oligonucleotide (e.g., an RNAi oligonucleotide) and the position of these nucleotide modifications can affect the properties of the oligonucleotide. For example, the oligonucleotide can be delivered in vivo by being conjugated to a lipid nanoparticle (LNP) or a similar carrier or being enclosed in a lipid nanoparticle (LNP) or a similar carrier. However, when an oligonucleotide is not protected by LNP or a similar carrier, it may be advantageous for at least some nucleotides to be modified. Therefore, in some embodiments, all or substantially all nucleotides of an oligonucleotide are modified. In some embodiments, more than half of the nucleotides are modified. In some embodiments, less than half of the nucleotides are modified. In some embodiments, the sugar moieties of all nucleotides constituting the oligonucleotide are modified at the 2' position. The modification can be reversible or irreversible. In some embodiments, oligonucleotides disclosed herein have the number and type of modified nucleotides sufficient to produce desired properties (e.g., protection for enzymatic degradation, the ability and / or thermodynamic stability of targeting desired cells after in vivo administration).

[0274] Sugar modification

[0275] In some embodiments, oligonucleotides described herein (e.g., RNAi oligonucleotides) include modified sugars. In some embodiments, modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, wherein, for example, one or more modifications occur at the 2', 3', 4' and / or 5' carbon positions of the sugar. In some embodiments, modified sugars may also include non-natural alternative carbon structures, such as those found in locked nucleic acids ("LNAs"; see, e.g., Koshkin et al. (1998) Tetrahedon 54:3607-30), unlocked nucleic acids ("UNA"; see, e.g., Snead et al. (2013) Mol. Ther-Nucl. Acids 2:e103) and bridged nucleic acids ("BNAs"; see, e.g., Imanishi & Obika (2002) Chem Commun. (Camb) 21:1653-59).

[0276] In some embodiments, the nucleotide modification in the sugar includes a 2'-modification. In some embodiments, the 2'-modification can be 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA) or 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe or 2'-MOE. In some embodiments, the modification in the sugar includes a modification of the sugar ring, which may include a modification of one or more carbons of the sugar ring. For example, the modification of the sugar of the nucleotide can include the 2'-oxygen of the sugar being connected to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen being connected to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking a 2'-carbon to 3'-carbon bond. In some embodiments, the modified nucleotide has a thiol group, for example, at the 4' position of the sugar.

[0277] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more). In some embodiments, the sense strand of the oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more). In some embodiments, the antisense strand of the oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0278] In some embodiments, all nucleotides of the sense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the antisense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the oligonucleotide (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotides comprise 2'-modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinose nucleic acids).

[0279] In some embodiments, the present disclosure provides oligonucleotides with different modification patterns.In some embodiments, the oligonucleotides herein comprise a sense strand with a modification pattern as set forth in the Examples and Sequence Listing and an antisense strand with a modification pattern as set forth in the Examples and Sequence Listing.

[0280] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise an antisense strand having nucleotides modified by 2'-F. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising nucleotides modified by 2'-F and 2'-OMe. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand having nucleotides modified by 2'-F. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand comprising nucleotides modified by 2'-F and 2'-OMe.

[0281] In some embodiments, the oligonucleotides described herein comprise a sense strand, and about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some embodiments, about 11% of the nucleotides of the sense strand comprise a 2'-fluoro modification. In some embodiments, the oligonucleotides described herein comprise an antisense strand, and about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 32% of the nucleotides of the antisense strand comprise a 2'-fluoro modification. In some embodiments, about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the nucleotides of the oligonucleotide comprise a 2'-fluoro modification. In some embodiments, about 19% of the nucleotides in the oligonucleotide comprise a 2'-fluoro modification.

[0282] As used herein, oligonucleotide numbering for the sense strand and the antisense strand, respectively, starts with "position 1" from the 5' end.

[0283] In some embodiments, one or more of positions 8, 9, 10, or 11 of the sense strand is modified with a 2'-F group. In some embodiments, one or more of positions 3, 8, 9, 10, 12, 13, and 17 of the sense strand is modified with a 2'-F group. In some embodiments, one or more of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand is modified with a 2'-F group. In some embodiments, one or more of positions 2, 3, 4, 5, 7, 8, 10, 14, 16, and 19 are modified with a 2'-F group. In some embodiments, the sugar moiety at each nucleotide at positions 1-7 and 12-20 in the sense strand is modified with 2'-OMe. In some embodiments, the sugar moiety at each nucleotide at positions 1-7, 12-27, and 31-36 in the sense strand is modified with 2'-OMe. In some embodiments, the sugar moiety at each of the nucleotides at positions 6, 9, 11-13, 15, 17, 18, and 20-22 in the sense strand is modified with 2'-OMe.

[0284] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0285] a) SEQ ID NO: 579 and 600, respectively;

[0286] b) SEQ ID NO: 580 and 601, respectively;

[0287] c) SEQ ID NOs: 595 and 616, respectively;

[0288] d) SEQ ID NO: 590 and 611, respectively;

[0289] e) SEQ ID NOs: 596 and 617, respectively;

[0290] f) SEQ ID NOs: 597 and 618, respectively;

[0291] g) SEQ ID NOs: 585 and 606, respectively;

[0292] h) SEQ ID NOs: 586 and 607, respectively; and

[0293] i) SEQ ID NO: 587 and 608, respectively,

[0294] wherein one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.

[0295] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0296] a) SEQ ID NO: 579 and 600, respectively;

[0297] b) SEQ ID NO: 580 and 601, respectively;

[0298] c) SEQ ID NOs: 590 and 611, respectively;

[0299] d) SEQ ID NOs: 597 and 618, respectively; and

[0300] e) SEQ ID NO: 586 and 607, respectively,

[0301] wherein one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.

[0302] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 2, 5, and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0303] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 1, 2, 5, and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0304] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 2, 4, 5, and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0305] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 1, 2, 3, 5, 7, and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0306] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0307] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 1, 2, 3, 5, 10 and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0308] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 10 and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0309] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 2, 3, 5, 7, 10 and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0310] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0311] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, 8, 10, 14, 16 and 19 of the antisense strand being modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0312] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 is modified with 2'-F.

[0313] In some embodiments, the oligonucleotides provided herein comprise an antisense strand, wherein the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 of the antisense strand is modified with 2'-OMe.

[0314] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0315] In some embodiments, the oligonucleotides provided herein comprise an antisense strand having a sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with a modification selected from the group consisting of 2'-fluoro (2'-F), 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0316] In some embodiments, the oligonucleotides provided herein comprise a sense strand, the sugar moieties at positions 8-11 of the sense strand are modified with 2'-F. In some embodiments, the oligonucleotides provided herein comprise a sense strand, the sugar moieties at positions 3, 8, 9, 10, 12, 13, and 17 of the sense strand are modified with 2'-F. In some embodiments, the oligonucleotides provided herein comprise a sense strand, the sugar moieties at positions 1-7 and 12-17 or 12-20 of the sense strand are modified with 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand, the sugar moieties at positions 1-7, 12-27, and 31-36 of the sense strand are modified with 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand, the sugar moiety of each nucleotide at positions 1-7 and 12-17 or 12-20 of the sense strand is modified by a modification selected from 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA) and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA). In some embodiments, the oligonucleotides provided herein comprise a sense strand, the sugar moieties at positions 1-2, 4-7, 11, 14-16 and 18-20 of the sense strand are modified by 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand, the sugar moiety of each nucleotide at positions 1-2, 4-7, 11, 14-16, and 18-20 of the sense strand being modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0317] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 of the sense strand that is modified with 2'-F.

[0318] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 of the sense strand modified with 2'-OMe.

[0319] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sense strand having a position 1, a position 2, a position 3, a position 4, a position 5, a position 6, a position 7, a position 8, a position 9, a position 10, a position 11, a position 12, a position 13, a position 14, a position 15, a position 16, a position 17, a position 18, a position 19, a position 20, a position 21, a position 22, a position 23, a position 24, a position 25, a position 26, a position 27, a position 28, a position 29, a position 30, a position 31, a position 32 The sugar moiety at position 32, position 33, position 34, position 35 or position 36 is modified with a modification selected from 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA) and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0320] In some embodiments, the oligonucleotides provided herein comprise a sense strand having position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53 3. The sugar moiety at position 34, position 35 or position 36 is modified with a modification selected from 2'-fluoro (2'-F), 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA) and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (2'-FANA).

[0321] 5'-terminal phosphate

[0322] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) include a sense strand and an antisense strand, wherein the antisense strand includes a 5'-terminal phosphate. In some embodiments, the 5'-terminal phosphate group of the RNAi oligonucleotide enhances the interaction with Ago2. However, oligonucleotides comprising a 5'-phosphate group may be easily degraded via phosphatases or other enzymes, which may limit their performance and / or bioavailability in vivo. In some embodiments, the oligonucleotides herein include 5' phosphate analogs resistant to such degradation. In some embodiments, the phosphate analogs are oxymethylphosphonates, vinylphosphonates or malonylphosphonates, or a combination thereof. In certain embodiments, the 5' end of the oligonucleotide chain is attached to a chemical moiety ("phosphate mimics") that simulates the electrostatic properties and spatial properties of a natural 5'-phosphate group. In some embodiments, the sense and antisense strands of the oligonucleotides include a nucleotide sequence selected from the group consisting of:

[0323] a) SEQ ID NO: 579 and 600, respectively;

[0324] b) SEQ ID NO: 580 and 601, respectively;

[0325] c) SEQ ID NOs: 595 and 616, respectively;

[0326] d) SEQ ID NO: 590 and 611, respectively;

[0327] e) SEQ ID NOs: 596 and 617, respectively;

[0328] f) SEQ ID NOs: 597 and 618, respectively;

[0329] g) SEQ ID NOs: 585 and 606, respectively;

[0330] h) SEQ ID NOs: 586 and 607, respectively; and

[0331] i) SEQ ID NO: 587 and 608, respectively,

[0332] wherein the oligonucleotide comprises a 5'-terminal phosphate, optionally a 5'-terminal phosphate analog.

[0333] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0334] a) SEQ ID NO: 579 and 600, respectively;

[0335] b) SEQ ID NO: 580 and 601, respectively;

[0336] c) SEQ ID NOs: 590 and 611, respectively;

[0337] d) SEQ ID NOs: 597 and 618, respectively; and

[0338] e) SEQ ID NO: 586 and 607, respectively,

[0339] wherein the oligonucleotide comprises a 5'-terminal phosphate, optionally a 5'-terminal phosphate analog.

[0340] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have phosphate analogs (referred to as "4'-phosphate analogs") at the 4'-carbon position of the sugar. See, for example, International Patent Application Publication No. WO 2018 / 045317. In some embodiments, the oligonucleotides herein include 4'-phosphate analogs at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bound to a sugar moiety (e.g., at its 4'-carbon) or an analog thereof. In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4'-carbon of a sugar moiety or its analog. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula –O–CH2–PO(OH)2, –O–CH2–PO(OR)2, or –O-CH2-PO(OH)(R), wherein R is independently selected from H, CH3, alkyl, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3. In some embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is 4'-oxymethylphosphonate.

[0341] In some embodiments, an oligonucleotide provided herein comprises an antisense strand comprising a 4'-phosphate analog at the 5'-terminal nucleotide, wherein the 5'-terminal nucleotide comprises the following structure:

[0342]

[0343] 4'-O-monomethylphosphonate-2'-O-methyluridine phosphorothioate [MePhosphonate-4O-mUs].

[0344] In some embodiments, an oligonucleotide provided herein comprises an antisense strand comprising a 4'-phosphate analog at the 5'-terminal nucleotide, wherein the 5'-terminal nucleotide comprises the following structure:

[0345]

[0346] 4'-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU].

[0347] When an internucleotide phosphorothioate bond is provided, chemistry 1a may be referred to as 4'-O-monomethylphosphonate-2'-O-methyluridine phosphorothioate [MePhosphonate-4O-mUs].

[0348] Modified internucleotide linkages

[0349] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) include modified internucleotide bonds. In some embodiments, phosphate modification or substitution produces an oligonucleotide comprising at least about 1 (e.g., at least 1, at least 2, at least 3, or at least 5) modified internucleotide bonds. In some embodiments, any oligonucleotide disclosed herein includes about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified internucleotide bonds. In some embodiments, any oligonucleotide disclosed herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide bonds.

[0350] The modified internucleotide linkage can be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, an alkylthiophosphonate linkage, an alkylthiophosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage, or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any oligonucleotide disclosed herein is a phosphorothioate linkage.

[0351] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) have a phosphorothioate bond between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotides described herein have a phosphorothioate bond between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the sense and antisense strands of the oligonucleotides comprise a nucleotide sequence selected from the group consisting of:

[0352] a) SEQ ID NO: 579 and 600, respectively;

[0353] b) SEQ ID NO: 580 and 601, respectively;

[0354] c) SEQ ID NOs: 595 and 616, respectively;

[0355] d) SEQ ID NO: 590 and 611, respectively;

[0356] e) SEQ ID NOs: 596 and 617, respectively;

[0357] f) SEQ ID NOs: 597 and 618, respectively;

[0358] g) SEQ ID NOs: 585 and 606, respectively;

[0359] h) SEQ ID NOs: 586 and 607, respectively; and

[0360] i) SEQ ID NO: 587 and 608, respectively,

[0361] wherein the oligonucleotide comprises modified internucleotide linkages.

[0362] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0363] a) SEQ ID NO: 579 and 600, respectively;

[0364] b) SEQ ID NO: 580 and 601, respectively;

[0365] c) SEQ ID NOs: 590 and 611, respectively;

[0366] d) SEQ ID NOs: 597 and 618, respectively; and

[0367] e) SEQ ID NO: 586 and 607, respectively,

[0368] wherein the oligonucleotide comprises modified internucleotide linkages.

[0369] Base modification

[0370] In some embodiments, oligonucleotides provided herein (e.g., RNAi oligonucleotides) include one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are connected at the 1' position of the nucleotide sugar moiety. In certain embodiments, modified nucleobases are nitrogenous bases. In some embodiments, modified nucleobases do not contain nitrogen atoms. See, for example, U.S. Patent Application Publication No. 2008 / 0274462. In some embodiments, modified nucleotides include universal bases. In some embodiments, modified nucleotides do not contain nucleobases (abasic). In some embodiments, the sense strand and antisense strand of an oligonucleotide include a nucleotide sequence selected from the group consisting of:

[0371] a) SEQ ID NO: 579 and 600, respectively;

[0372] b) SEQ ID NO: 580 and 601, respectively;

[0373] c) SEQ ID NOs: 595 and 616, respectively;

[0374] d) SEQ ID NO: 590 and 611, respectively;

[0375] e) SEQ ID NOs: 596 and 617, respectively;

[0376] f) SEQ ID NOs: 597 and 618, respectively;

[0377] g) SEQ ID NOs: 585 and 606, respectively;

[0378] h) SEQ ID NOs: 586 and 607, respectively; and

[0379] i) SEQ ID NO: 587 and 608, respectively,

[0380] wherein the oligonucleotide comprises one or more modified nucleobases.

[0381] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0382] a) SEQ ID NO: 579 and 600, respectively;

[0383] b) SEQ ID NO: 580 and 601, respectively;

[0384] c) SEQ ID NOs: 590 and 611, respectively;

[0385] d) SEQ ID NOs: 597 and 618, respectively; and

[0386] e) SEQ ID NO: 586 and 607, respectively,

[0387] wherein the oligonucleotide comprises one or more modified nucleobases.

[0388] In some embodiments, the universal base is a heterocyclic moiety that is located at the 1' position of the nucleotide sugar moiety in the modified nucleotide, or at an equivalent position in a nucleotide sugar moiety substitution, such that when present in a duplex, it can be placed opposite more than one type of base without substantially altering the structure of the duplex. In some embodiments, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is fully complementary to the target nucleic acid (e.g., TMPRSS6 mRNA), and the T of the duplex is m In some embodiments, when compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid, and the T of the duplex is m Higher than the duplex formed with a nucleic acid containing the mismatched base.

[0389] Non-limiting examples of universal binding nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al. (1995) Nucleic Acids Res. 23:4363-4370; Loakes et al. (1995) Nucleic Acids Res. 23:2361-66; and Loakes & Brown (1994) Nucleic Acids Res. 22:4039-43).

[0390] Targeting ligands

[0391] In some embodiments, it is desirable to target the oligonucleotides provided herein (e.g., RNAi oligonucleotides) to one or more cells or cell types, tissues, organs, or anatomical regions or compartments. Such a strategy can help to avoid adverse effects on the treated organism and / or avoid excessive loss of oligonucleotides to cells, tissues, organs, or anatomical regions or compartments that will not benefit from the oligonucleotides or their effects (e.g., inhibition or reduction of TMPRSS6 expression). Therefore, in some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) are modified to facilitate targeting and / or delivery to specific cells or cell types, tissues, organs, or anatomical regions or compartments (e.g., to promote delivery of oligonucleotides to the liver). In some embodiments, the oligonucleotides comprise at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more targeting ligands. In some embodiments, the sense strand and antisense strand of the oligonucleotides comprise a nucleotide sequence selected from the group consisting of:

[0392] a) SEQ ID NO: 579 and 600, respectively;

[0393] b) SEQ ID NO: 580 and 601, respectively;

[0394] c) SEQ ID NOs: 595 and 616, respectively;

[0395] d) SEQ ID NO: 590 and 611, respectively;

[0396] e) SEQ ID NOs: 596 and 617, respectively;

[0397] f) SEQ ID NOs: 597 and 618, respectively;

[0398] g) SEQ ID NOs: 585 and 606, respectively;

[0399] h) SEQ ID NOs: 586 and 607, respectively; and

[0400] i) SEQ ID NO: 587 and 608, respectively,

[0401] wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide.

[0402] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0403] a) SEQ ID NO: 579 and 600, respectively;

[0404] b) SEQ ID NO: 580 and 601, respectively;

[0405] c) SEQ ID NOs: 590 and 611, respectively;

[0406] d) SEQ ID NOs: 597 and 618, respectively; and

[0407] e) SEQ ID NO: 586 and 607, respectively,

[0408] wherein the oligonucleotide comprises a targeting ligand conjugated to at least one nucleotide.

[0409] In some embodiments, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a peptide, a polypeptide, a protein or a portion of a protein (e.g., an antibody or antibody fragment) or a lipid. In certain embodiments, the targeting ligand is a carbohydrate comprising at least one GalNAc moiety.

[0410] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide (e.g., RNAi oligonucleotide) provided herein are each conjugated to a separate targeting ligand (e.g., a GalNAc moiety). In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, a targeting ligand is conjugated to 2 to 4 nucleotides at either end of a sense strand or antisense strand (e.g., a targeting ligand is conjugated to an overhang or extension of 2 to 4 nucleotides at the 5' or 3' end of a sense strand or antisense strand), such that the targeting ligand is similar to the bristles of a toothbrush, and the oligonucleotide is similar to a toothbrush. For example, an oligonucleotide may include a stem-loop at the 5' or 3' end of a sense strand, and 1, 2, 3, or 4 nucleotides of the loop of the stem may be conjugated to a targeting ligand individually. In some embodiments, the oligonucleotides provided by the present disclosure (e.g., RNAi oligonucleotides) comprise a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a triloop or a tetraloop, and wherein the 3 or 4 nucleotides constituting the triloop or the tetraloop are conjugated to a targeting ligand, respectively. In some embodiments, the oligonucleotides provided by the present disclosure (e.g., RNAi oligonucleotides) comprise a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a tetraloop, and wherein the 3 nucleotides of the tetraloop are conjugated to a targeting ligand, respectively.

[0411] GalNAc is a high affinity carbohydrate ligand for the asialoglycoprotein receptor (ASGPR), which is primarily expressed on the surface of hepatocytes and plays a major role in binding, internalization, and subsequent clearance of circulating glycoproteins (asialoglycoproteins) containing terminal galactose or GalNAc residues. Conjugation (indirect or direct) of the GalNAc moiety to the oligonucleotides of the present disclosure can be used to target these oligonucleotides to the ASGPR expressed on cells. In some embodiments, the oligonucleotides of the present disclosure (e.g., RNAi oligonucleotides) are conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotides to the ASGPR expressed on human liver cells (e.g., human hepatocytes). In some embodiments, the GalNAc moieties target the oligonucleotides to the liver.

[0412] In some embodiments, the oligonucleotides of the present disclosure (e.g., RNAi oligonucleotides) are directly or indirectly conjugated to a monovalent GalNAc moiety. In some embodiments, the oligonucleotide is directly or indirectly conjugated to more than one monovalent GalNAc (i.e., conjugated to 2, 3, or 4 monovalent GalNAc moieties, and generally conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotide is conjugated to one or more divalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties. In some embodiments, the divalent, trivalent, or tetravalent GalNAc moieties are conjugated to the oligonucleotide via a branched connector. In some embodiments, the monovalent GalNAc moiety is conjugated to the first nucleotide, and the divalent, trivalent, or tetravalent GalNAc moieties are conjugated to the second nucleotide via a branched connector.

[0413] In some embodiments, one (1) or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide described herein (e.g., an RNAi oligonucleotide) are each conjugated to a GalNAc moiety. In some embodiments, two (2) to four (4) nucleotides of a tetracyclic ring are each conjugated to a separate GalNAc moiety. In some embodiments, one (1) to three (3) nucleotides of a tricyclic ring are each conjugated to a separate GalNAc moiety. In some embodiments, a targeting ligand is conjugated to two (2) to four (4) nucleotides at either end of the sense strand or antisense strand (e.g., a ligand is conjugated to an overhang or extension of two (2) to four (4) nucleotides at the 5' or 3' end of the sense strand or antisense strand), such that the GalNAc moiety is similar to the bristles of a toothbrush, and the oligonucleotide is similar to a toothbrush. In some embodiments, the GalNAc moiety is conjugated to the nucleotides of the sense strand. For example, three (3) or four (4) GalNAc moieties can be conjugated to nucleotides in the four loops of the sense strand, wherein each GalNAc moiety is conjugated to one (1) nucleotide.

[0414] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a tetracycle, wherein the tetracycle (tetraLp) is any combination of adenine (A) and guanine (G) nucleotides. In some embodiments, the tetracycle (tetraLp) comprises a monovalent GalNAc moiety attached to any one or more guanine (G) nucleotides of the tetracycle via any linker described herein, as shown in the following Chemical Formula 2 (X = heteroatom):

[0415]

[0416] It should be understood that when included in an oligonucleotide, Formula 2 can be covalently linked to adjacent nucleotides. In some embodiments, the tetraloop (tetraLp) comprises a monovalent GalNAc moiety attached to any one or more guanine (G) nucleotides of the tetraloop via any linker described herein, as shown in the following Formula 2a (X = heteroatom):

[0417]

[0418]

[0419] In some embodiments, the tetraloop (tetraLp) has a monovalent GalNAc attached to any one or more adenine (A) nucleotides of the tetraloop via any linker described herein, as shown in the following Chemical Formula 3 (X=heteroatom):

[0420]

[0421] It should be understood that when included in an oligonucleotide, Formula 3 can be covalently linked to adjacent nucleotides. In some embodiments, the tetraloop (tetraLp) has a monovalent GalNAc attached to any one or more adenine (A) nucleotides of the tetraloop via any linker described herein, as shown in the following Formula 3a (X = heteroatom):

[0422]

[0423] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a monovalent GalNAc moiety attached to a guanine (G) nucleotide, referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc, as shown below (Chemical Formula 4):

[0424] (Chemical formula 4)

[0425] It should be understood that when included in an oligonucleotide, Formula 4 may be linked to adjacent nucleotides, such as shown in the following Formula 4a:

[0426]

[0427] In some embodiments, the oligonucleotides herein comprise a monovalent GalNAc moiety attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as shown below (Chemical Formula 5):

[0428]

[0429] It should be understood that when included in an oligonucleotide, Formula 5 may be linked to adjacent nucleotides, such as shown in the following Formula 5a:

[0430]

[0431] An example of such conjugation is shown below for a loop containing the nucleotide sequence GAAA from 5' to 3' (Formula 6) (L = linker, X = heteroatom). For example, such a loop can be present at positions 27-30 of the sense strand provided herein. In the formula, Used to describe the connection point to the oligonucleotide chain (Chemical Formula 6).

[0432]

[0433] Another example of such conjugation is shown below for a loop containing the nucleotide sequence GAAA from 5' to 3' (Formula 6a) (L = linker, X = heteroatom). For example, such a loop can be present at positions 27-30 of the sense strand provided herein. In the formula, Used to describe the point of attachment to an oligonucleotide chain.

[0434]

[0435] The targeting ligand can be connected to the nucleotide using an appropriate method or chemistry (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotides constituting the oligonucleotides (e.g., RNAi oligonucleotides) herein using a click connector. In some embodiments, the targeting ligand is conjugated to the nucleotides of any of the oligonucleotides described herein using an acetal-based connector. Acetal-based connectors are disclosed, for example, in an international patent application with publication number WO2016 / 100401. In some embodiments, the connector is an unstable connector. However, in other embodiments, the connector is stable. An example of a ring from 5' to 3' containing the nucleotide GAAA is shown below, in which the GalNAc moiety is attached to the nucleotides of the ring using an acetal connector (Chemical Formula 7 and Chemical Formula 8). For example, such a ring can be present at positions 27-30 of any sense strand. In the chemical formula, It is the connection point to the oligonucleotide chain (Chemical Formula 7 and Chemical Formula 8).

[0436]

[0437]

[0438] An example of a loop comprising nucleotides GAAA from 5' to 3' is shown below, wherein the GalNAc moiety is attached to the nucleotides of the loop using an acetal linker (Formula 7a and Formula 8a). For example, such a loop can be present at positions 27-30 of either sense strand. In the formula, is the connection point to the oligonucleotide chain (Chemical Formula 7a and Chemical Formula 8a):

[0439]

[0440]

[0441] As mentioned, the targeting ligand can be connected to the nucleotide using various appropriate methods or chemical synthesis techniques (e.g., click chemistry). In some embodiments, the targeting ligand is conjugated to the nucleotide using a click connector. In some embodiments, the targeting ligand is conjugated to the nucleotide of any oligonucleotide described herein using an acetal-based connector. Acetal-based connectors are disclosed, for example, in the international patent application publication number WO 2016 / 100401. In some embodiments, the connector is an unstable connector. However, in other embodiments, the connector is a stable connector.

[0442] In some embodiments, an oligonucleotide herein (eg, an RNAi oligonucleotide) does not have GalNAc conjugated thereto.

[0443] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0444] a) SEQ ID NO: 579 and 600, respectively;

[0445] b) SEQ ID NO: 580 and 601, respectively;

[0446] c) SEQ ID NOs: 595 and 616, respectively;

[0447] d) SEQ ID NO: 590 and 611, respectively;

[0448] e) SEQ ID NOs: 596 and 617, respectively;

[0449] f) SEQ ID NOs: 597 and 618, respectively;

[0450] g) SEQ ID NOs: 585 and 606, respectively;

[0451] h) SEQ ID NOs: 586 and 607, respectively; and

[0452] i) SEQ ID NO: 587 and 608, respectively,

[0453] wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0454] In some embodiments, the sense strand and the antisense strand of the oligonucleotide comprise a nucleotide sequence selected from the group consisting of:

[0455] a) SEQ ID NO: 579 and 600, respectively;

[0456] b) SEQ ID NO: 580 and 601, respectively;

[0457] c) SEQ ID NOs: 590 and 611, respectively;

[0458] d) SEQ ID NOs: 597 and 618, respectively; and

[0459] e) SEQ ID NO: 586 and 607, respectively,

[0460] Wherein the oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide. Exemplary oligonucleotides for reducing TMPRSS6 expression

[0461] In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure include a sense strand and an antisense strand, wherein all nucleotides constituting the sense strand and the antisense strand are modified, wherein the antisense strand includes a complementary region complementary to the TMPRSS6 mRNA target sequence of any one of SEQ ID NOs: 661-852, and wherein the length of the complementary region is at least 15 consecutive nucleotides. In some embodiments, the 5'-terminal nucleotide of the antisense strand includes 4'-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU], as described herein. In some embodiments, the 5'-terminal nucleotide of the antisense strand includes a phosphorothioate bond. In some embodiments, the antisense strand and the sense strand include one or more 2'-fluoro (2'-F) and 2'-O-methyl (2'-OMe) modified nucleotides and at least one phosphorothioate bond. In some embodiments, the antisense strand comprises four (4) phosphorothioate bonds, while the sense strand comprises one (1) phosphorothioate bond. In some embodiments, the antisense strand comprises five (5) phosphorothioate bonds, while the sense strand comprises one (1) phosphorothioate bond.

[0462] In some embodiments, an oligonucleotide provided herein (eg, an RNAi oligonucleotide) comprises a sense strand having a sequence of any one of SEQ ID NOs: 193-384 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 385-576.

[0463] In some embodiments, an oligonucleotide provided herein (eg, an RNAi oligonucleotide) comprises a sense strand having a sequence of any one of SEQ ID NOs: 577-597 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 598-618.

[0464] In some embodiments, an oligonucleotide provided herein (eg, an RNAi oligonucleotide) comprises a sense strand having a sequence of any one of SEQ ID NOs: 619-639 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 640-660.

[0465] In some embodiments, the oligonucleotides provided herein for reducing TMPRSS6 expression (e.g., RNAi oligonucleotides) comprise:

[0466] a sense strand of 36 nucleotides comprising 2'-F modified nucleotides at positions 8-11, 2'-OMe modified nucleotides at positions 1-7, 12-27 and 31-36, GalNAc conjugated nucleotides at positions 28, 29 and 30; and a phosphorothioate bond between positions 1 and 2;

[0467] The invention relates to an antisense strand of 22 nucleotides comprising 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10 and 14, 2'-OMe at positions 1, 6, 8, 9, 11-13 and 15-22, phosphorothioate bonds between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21 and positions 21 and 22, and a 5'-terminal nucleotide at position 1 comprising a 4'-phosphate analog, optionally wherein the 5'-terminal nucleotide comprises a 4'- O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand form a duplex region with positions 1-20 of the sense strand, wherein positions 21-36 of the sense strand form a stem-loop, wherein positions 27-30 form a loop of the stem-loop, optionally wherein positions 27-30 comprise a tetraloop, wherein positions 21 and 22 of the antisense strand comprise overhangs, and wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0468] a) SEQ ID NO: 579 and 600, respectively;

[0469] b) SEQ ID NO: 580 and 601, respectively;

[0470] c) SEQ ID NOs: 595 and 616, respectively;

[0471] d) SEQ ID NO: 590 and 611, respectively;

[0472] e) SEQ ID NOs: 596 and 617, respectively;

[0473] f) SEQ ID NOs: 597 and 618, respectively;

[0474] g) SEQ ID NOs: 585 and 606, respectively;

[0475] h) SEQ ID NOs: 586 and 607, respectively; and

[0476] i) are SEQ ID NOs: 587 and 608, respectively.

[0477] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises:

[0478] a sense strand of 36 nucleotides comprising 2'-F modified nucleotides at positions 8-11, 2'-OMe modified nucleotides at positions 1-7, 12-27 and 31-36, GalNAc conjugated nucleotides at positions 28, 29 and 30; and a phosphorothioate bond between positions 1 and 2;

[0479] The invention relates to an antisense strand of 22 nucleotides comprising 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10 and 14, 2'-OMe at positions 1, 6, 8, 9, 11-13 and 15-22, phosphorothioate bonds between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21 and positions 21 and 22, and a 5'-terminal nucleotide at position 1 comprising a 4'-phosphate analog, optionally wherein the 5'-terminal nucleotide comprises a 4'- O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand form a duplex region with positions 1-20 of the sense strand, wherein positions 21-36 of the sense strand form a stem-loop, wherein positions 27-30 form a loop of the stem-loop, optionally wherein positions 27-30 comprise a tetraloop, wherein positions 21 and 22 of the antisense strand comprise overhangs, and wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0480] a) SEQ ID NO: 579 and 600, respectively;

[0481] b) SEQ ID NO: 580 and 601, respectively;

[0482] c) SEQ ID NOs: 590 and 611, respectively;

[0483] d) SEQ ID NOs: 597 and 618, respectively; and

[0484] e) are SEQ ID NOs: 586 and 607, respectively.

[0485] In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 579, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 600. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 580, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 601. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 590, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 611. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 597, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 618. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 586, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 607.

[0486] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 184; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0487] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 181; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0488] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 158; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0489] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 134; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0490] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 102; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0491] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 184; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0492] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 181; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0493] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 158; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0494] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 134; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0495] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 102; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0496] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 184; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the complementary region that is complementary to the antisense strand is as shown in SEQ ID NO: 844, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0497] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 181; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the complementary region that is complementary to the antisense strand is as shown in SEQ ID NO: 841, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0498] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 158; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the complementary region that is complementary to the antisense strand is as shown in SEQ ID NO: 818, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0499] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 134; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the complementary region that is complementary to the antisense strand is as shown in SEQ ID NO: 794, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0500] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region that is complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 102; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region that is complementary to the antisense strand, wherein the complementary region that is complementary to the antisense strand is as shown in SEQ ID NO: 762, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0501] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 184; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is as shown in SEQ ID NO: 844, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0502] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 181; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is as shown in SEQ ID NO: 841, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0503] In some embodiments, an oligonucleotide targeting TMPRSS6 (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 158; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is as shown in SEQ ID NO: 818, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0504] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 134; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is as shown in SEQ ID NO: 794, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0505] In some embodiments, an oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression (e.g., an RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 102; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is as shown in SEQ ID NO: 762, wherein the stem-loop is represented as S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0506] In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand according to:

[0507] Sense strand: 5'-mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX- mX-mX-mX-mX-[ademX-GalNAc]-[ademX-GalNAc]-[ademX-GalNAc]-mX-mX-mX-mX-mX-mX-3';

[0508] Hybridize to:

[0509] Antisense strand: 5'-[MePhosphonate-4O-mX]-S-fX-S-fX-S-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX-S-mX-S-mX-3';

[0510] Where mX = 2'-O-methyl modified nucleotide,

[0511] fX = 2'-fluoro-modified nucleotide,

[0512] -S- = phosphorothioate bond,

[0513] - = phosphodiester bond,

[0514] [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and

[0515] ademX-GalNAc = GalNAc attached to a nucleotide.

[0516] In some embodiments, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mGs][mG][mU][mG][mC][mU][mA][fC][fU][fC][fU][mG][mG][mU][mA][mU][mU][mU][mC][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3'(SEQ ID NO: 1). ID NO:621) and all modifications, and wherein the antisense strand comprises the sequence of 5'[MePhosphonate-4O-mUs][fGs][fAs][fA][fA][mU][fA][mC][mC][fA][mG][mA][mG][fU][mA][mG][mC][mA][mCs][mGs][mG]-3' (SEQ ID NO:642) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0517] GalNAc-modified adenine nucleotides.

[0518] In some embodiments, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5' [mGs][mC][mU][mA][mC][mU][mC][fU][fG][fG][fU][mA][mU][mU][mC][mC][mU][mA][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:622) and all modifications, and wherein the antisense strand comprises the sequence of 5'[MePhosphonate-4O-mUs][fUs][fAs][fG][fG][mA][fA][mA][mU][fA][mC][mC][mA][fG][mA][mG][mU][mA][mG][mCs][mGs][mG]-3' (SEQ ID NO:643) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0519] GalNAc-modified adenine nucleotides.

[0520] In some embodiments, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mCs][mU][mC][mA][mC][mC][mU][fG][fC][fU][fU][mC][mU][mC][mU][mG][mG][mU][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3'(SEQ ID NO: 1). ID NO:632) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fAs][fCs][fC][fA][mG][fA][mA][mG][fA][mA][mG][mC][fA][mG][mG][mU][mG][mA][mGs][mGs][mG]-3' (SEQ ID NO:653) and all modifications, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = thiophosphate, and wherein ademA-GalNAc =

[0521] GalNAc-modified adenine nucleotides.

[0522] In some embodiments, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mAs][mG][mU][mG][mU][mG][mA][fA][fA][fG][fA][mC][mA][mU][mA][mG][mC][mU][mG][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]3'(SEQ ID NO: 1). ID NO:639) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fCs][fAs][fG][fC][mU][fA][mU][mG][fU][mC][mU][mU][fU][mC][mA][mC][mA][mC][mUs][mGs][mG]-3' (SEQ ID NO:660) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein ademA-GalNAc=

[0523] GalNAc-modified adenine nucleotides.

[0524] In some embodiments, the present disclosure provides an RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mGs][mG][mG][mU][mG][mC][mA][fC][fU][fA][fU][mG][mG][mC][mU][mU][mG][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]3'(SEQ ID NO: 1). ID NO:628) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fUs][fAs][fC][fA][mA][fG][mC][mC][fA][mU][mA][mG][fU][mG][mC][mA][mC][mCs][mGs][mG]-3' (SEQ ID NO:649) and all modifications, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = thiophosphate, and wherein ademA-GalNAc = GalNAc-modified adenine nucleotide.

[0525] In some embodiments, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide) for reducing TMPRSS6 expression, wherein the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising a nucleotide sequence selected from the group consisting of:

[0526] a) SEQ ID NO: 621 and 642, respectively;

[0527] b) SEQ ID NO: 622 and 643, respectively;

[0528] c) SEQ ID NOs: 637 and 658, respectively;

[0529] d) SEQ ID NOs: 632 and 653, respectively;

[0530] e) SEQ ID NOs: 638 and 659, respectively;

[0531] f) SEQ ID NOs: 639 and 660, respectively;

[0532] g) SEQ ID NOs: 627 and 648, respectively;

[0533] h) SEQ ID NOs: 628 and 649, respectively; and

[0534] i) are SEQ ID NOs: 629 and 650, respectively.

[0535] In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 621, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 642. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 622, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 643. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 632, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 653. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 639, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 660. In some embodiments, the oligonucleotides targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprise a sense strand and an antisense strand, the sense strand comprising the nucleotide sequence shown in SEQ ID NO: 628, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 649.

[0536] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 621, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 642, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 643. Fig.14A The conjugate forms of the structures shown.

[0537] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 621, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 642, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 643. Fig. 10A -The form of the conjugate of the structure shown in B.

[0538] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 632, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 653, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 654. Fig. 14B The conjugate forms of the structures shown.

[0539] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 632, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 653, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 654. Fig.11A -The form of the conjugate of the structure shown in B.

[0540] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 639, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 660, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 661. Fig. 14C The conjugate forms of the structures shown.

[0541] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 639, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 660, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 661. Fig. 12A -The form of the conjugate of the structure shown in B.

[0542] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 628, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 649, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 649. Fig.14D The conjugate forms of the structures shown.

[0543] In some embodiments, the oligonucleotide targeting TMPRSS6 for reducing TMPRSS6 expression provided by the present disclosure comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 628, the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 649, and the antisense strand comprising a complementary region complementary to a TMPRSS6 RNA transcript, wherein the RNAi is a nucleotide sequence as shown in SEQ ID NO: 649. Fig.13A -The form of the conjugate of the structure shown in B.

[0544] preparation

[0545] A variety of preparations (e.g., pharmaceutical preparations) for oligonucleotide applications have been developed. For example, oligonucleotides (e.g., RNAi oligonucleotides) can be delivered to a subject or a cell environment using a preparation that minimizes degradation, promotes delivery and / or uptake, or provides another beneficial property for the oligonucleotides in the preparation. In some embodiments, compositions comprising oligonucleotides (e.g., RNAi oligonucleotides) that reduce TMPRSS6 expression are provided herein. Such compositions can be appropriately formulated so that when applied to a subject, whether it is administered to the direct environment of the target cell or systemically, a sufficient portion of the oligonucleotides enters the cell to reduce TMPRSS6 expression. As disclosed herein, any type of suitable oligonucleotide preparation can be used to deliver oligonucleotides to reduce TMPRSS6. In some embodiments, the oligonucleotides are formulated in a buffer solution such as a phosphate buffered saline solution, a liposome, a micellar structure, and a shell. Any oligonucleotide described herein can be provided not only as a nucleic acid, but also in the form of a pharmaceutically acceptable salt.

[0546] Oligonucleotide preparations with cationic lipids can be used to promote the transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.) or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

[0547] Thus, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere or a nanoparticle, or can be otherwise formulated for administration to a cell, a tissue, an organ or a body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd ed., Pharmaceutical Press, 2013).

[0548] In some embodiments, the formulations herein include adjuvants. In some embodiments, the adjuvants provide improved stability, improved absorption, improved solubility, and / or therapeutic enhancement to the active ingredient conferred by the composition. In some embodiments, the adjuvant is a buffer (e.g., sodium citrate, sodium phosphate, tris alkali, or sodium hydroxide) or a vehicle (e.g., a buffer solution, vaseline, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotides are lyophilized to extend their shelf life and then made into a solution before use (e.g., administration to a subject). Therefore, the adjuvant in the composition comprising any of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrrolidone) or a collapse temperature regulator (e.g., dextran, Ficoll TM or gelatin).

[0549] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0550] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. Sterile injection solutions can be prepared as follows: the desired amount of oligonucleotides and one or a combination of the ingredients listed above are introduced into a selected solvent as required, followed by filtration sterilization.

[0551] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an RNAi oligonucleotide for reducing TMPRSS6 expression), although the percentage of active ingredient may be between about 1% and about 80% or more by weight or volume of the total composition. One skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore, a variety of dosages and treatment regimens may be desirable.

[0552] How to use

[0553] Reduction of TMPRSS6 expression

[0554] In some embodiments, the present disclosure provides methods for contacting a cell or cell population with or delivering an effective amount of an oligonucleotide provided herein (e.g., an RNAi oligonucleotide) to reduce TMPRSS6 expression. In some embodiments, the reduction in TMPRSS6 expression is determined by measuring the reduction in the amount or level of TMPRSS6 mRNA, membrane-type serine protease-2 protein, or membrane-type serine protease-2 activity in the cell. These methods include those described herein and known to those of ordinary skill in the art.

[0555] The methods provided herein can be used for any suitable cell type. In some embodiments, the cell is any cell (e.g., a hepatocyte) expressing TMPRSS6 mRNA. In some embodiments, the cell is a primary cell obtained from a subject. In some embodiments, the primary cell has undergone a limited number of passages so that the cell substantially maintains its natural phenotypic properties. In some embodiments, the cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., it can be delivered to a cell in culture or an organism in which the cell is located).

[0556] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) are delivered to cells or cell colonies using nucleic acid delivery methods known in the art, including but not limited to injection of a solution containing an oligonucleotide, bombardment with particles covered with an oligonucleotide, exposing the cell or cell colony to a solution containing an oligonucleotide, or electroporating the cell membrane in the presence of an oligonucleotide. Other methods known in the art for delivering oligonucleotides to cells can be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, etc.

[0557] In some embodiments, the reduction in TMPRSS6 expression is determined by evaluating one or more molecules, properties or characteristics of cells or cell populations associated with TMPRSS6 expression, or by evaluating a molecule (e.g., TMPRSS6 mRNA or membrane-type serine protease-2 protein) that directly indicates TMPRSS6 expression in a cell or cell population. Determined by the determination or technology. In some embodiments, the degree to which the oligonucleotides provided herein reduce TMPRSS6 expression is evaluated by comparing TMPRSS6 expression in cells or cell populations contacted with oligonucleotides with appropriate controls (e.g., appropriate cells or cell populations not contacted with the oligonucleotides or contacted with control oligonucleotides). In some embodiments, the control amount or level of TMPRSS6 expression in the control cells or cell populations is predetermined so that the control amount or level does not need to be measured each time the determination or technology is performed. The predetermined level or value can take a variety of forms. In some embodiments, the predetermined level or value can be a single cutoff value, such as a median or mean value.

[0558] In some embodiments, contacting or delivering an oligonucleotide described herein (e.g., an RNAi oligonucleotide) to a cell or cell population results in a decrease in TMPRSS6 expression in a cell or cell population that has not been contacted with the oligonucleotide or has been contacted with a control oligonucleotide. In some embodiments, the decrease in TMPRSS6 expression is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less relative to a control amount or level of TMPRSS6 expression. In some embodiments, the control amount or level of TMPRSS6 expression is the amount or level of TMPRSS6 mRNA and / or membrane-type serine protease-2 protein in a cell or cell population that has not been contacted with an oligonucleotide herein. In some embodiments, TMPRSS6 mRNA expression is measured using methods known in the art. In some embodiments, TMPRSS6 mRNA expression is measured by qPCR. In some embodiments, TMPRSS6 protein expression is measured using methods known in the art. In some embodiments, TMPRSS6 protein expression is measured by ELISA. In some embodiments, TMPRSS6 protein expression is measured by Western blotting. In some embodiments, the effect of delivering the oligonucleotides herein to cells or cell populations according to the methods herein is evaluated after any limited period of time or amount of time (e.g., minutes, hours, days, weeks, months). For example, in some embodiments, TMPRSS6 expression is determined in a cell or cell population at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours after contacting or delivering an oligonucleotide to a cell or cell population; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or more. In some embodiments, TMPRSS6 expression is determined in a cell or cell population at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or more after contacting or delivering an oligonucleotide to a cell or cell population.

[0559] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) are delivered in the form of transgenics that are engineered to express the oligonucleotides or the strands that constitute the oligonucleotides in cells (e.g., sense and antisense strands thereof). In some embodiments, the oligonucleotides herein are delivered using transgenics that are engineered to express any oligonucleotide disclosed herein. Transgenics can be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNA). In some embodiments, transgenics can be injected directly into a subject.

[0560] Treatment

[0561] The present disclosure provides oligonucleotides (e.g., RNAi oligonucleotides) for use as medicaments, in particular for use in methods for treating diseases, disorders, and conditions associated with hepcidin deficiency or inhibition. The present disclosure also provides oligonucleotides for or suitable for use in treating subjects who would benefit from reducing TMPRSS6 expression (e.g., people suffering from diseases, disorders, or conditions associated with hepcidin deficiency or inhibition). In some aspects, the present disclosure provides oligonucleotides for or suitable for use in treating subjects suffering from diseases, disorders, or conditions associated with hepcidin deficiency or inhibition. In some embodiments, the subject in need of reducing TMPRSS6 suffers from an iron accumulation disease, disorder, or condition, or is at risk of suffering from an iron accumulation disease, disorder, or condition. In some embodiments, compositions, compounds, and methods described herein for reducing iron levels in an individual are provided. In some embodiments, iron accumulation is due to a disease, disorder, or condition of the subject. In some embodiments, the disease, disorder, and / or condition is hemochromatosis, such as hereditary hemochromatosis. In some embodiments, the disease, disorder, and / or condition is β-thalassemia. In some embodiments, the disease, disorder, and / or condition is polycythemia vera. The present disclosure also provides oligonucleotides for use or suitable for use in the preparation of drugs or pharmaceutical compositions for treating diseases, disorders or conditions associated with hepcidin deficiency or inhibition. In some embodiments, the oligonucleotides for use or suitable for use target TMPRSS6 mRNA and reduce TMPRSS6 expression (e.g., via RNAi pathway). In some embodiments, the oligonucleotides for use or suitable for use target TMPRSS6 mRNA and reduce the amount or level of TMPRSS6 mRNA, membrane serine protease-2 protein and / or TMPRSS6 activity.

[0562] The present disclosure also provides methods for treating subjects suffering from, suspected of suffering from, or at risk of suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition using the oligonucleotides provided herein. In some aspects, the present disclosure provides methods for treating a disease, disorder, or condition associated with hepcidin deficiency or inhibition or attenuating its onset or progression using the oligonucleotides provided herein. In other aspects, the present disclosure provides methods for achieving one or more therapeutic benefits in subjects suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition using the oligonucleotides provided herein. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of any one or more of the oligonucleotides provided herein. In some embodiments, treatment includes reducing TMPRSS6 expression. In some embodiments, the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically.

[0563] In some embodiments of the methods herein, one or more oligonucleotides herein (e.g., RNAi oligonucleotides) or a pharmaceutical composition comprising one or more oligonucleotides are administered to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition, such that TMPRSS6 expression in the subject is reduced, thereby treating the subject. In some embodiments, the amount or level of TMPRSS6 mRNA in the subject is reduced. In some embodiments, the amount or level of membrane-type serine protease-2 protein in the subject is reduced. In some embodiments, the amount or level of membrane-type serine protease-2 activity in the subject is reduced.

[0564] In some embodiments of the methods herein, an oligonucleotide (e.g., an RNAi oligonucleotide) provided herein or a pharmaceutical composition comprising the oligonucleotide is administered to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition such that TMPRSS6 expression in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99%, compared to TMPRSS6 expression prior to administration of one or more oligonucleotides or pharmaceutical compositions. In some embodiments, the expression of TMPRSS6 in a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the expression of TMPRSS6 in a subject (e.g., a reference or control subject) that did not receive the one or more oligonucleotides or pharmaceutical compositions or received one or more control oligonucleotides, pharmaceutical compositions, or treatments.

[0565] In some embodiments of the methods herein, one or more oligonucleotides herein (e.g., RNAi oligonucleotides) or pharmaceutical compositions comprising the one or more oligonucleotides are administered to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition such that the amount or level of TMPRSS6 mRNA in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99%, compared to the amount or level of TMPRSS6 mRNA before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of TMPRSS6 mRNA in a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of TMPRSS6 mRNA in a subject (e.g., a reference or control subject) that did not receive the one or more oligonucleotides or pharmaceutical compositions or received one or more control oligonucleotides, pharmaceutical compositions, or treatments.

[0566] In some embodiments of the methods herein, one or more oligonucleotides herein or pharmaceutical compositions comprising the one or more oligonucleotides are administered to a subject suffering from a disease, disorder or condition associated with hepcidin deficiency or inhibition such that the amount or level of serpin-2 protein in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more than 99% compared to the amount or level of serpin-2 protein before administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of serpin-2 protein in a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of serpin-2 protein in a subject (e.g., a reference or control subject) that has not received the one or more oligonucleotides or pharmaceutical compositions or received one or more control oligonucleotides or pharmaceutical compositions or treatments.

[0567] In some embodiments of the methods herein, one or more oligonucleotides herein (e.g., RNAi oligonucleotides) or pharmaceutical compositions comprising the one or more oligonucleotides are administered to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition such that the amount or level of TMPRSS6 gene activity / expression in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99%, compared to the amount or level of TMPRSS6 activity prior to administration of the oligonucleotide or pharmaceutical composition. In some embodiments, the amount or level of TMPRSS6 activity in a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the amount or level of TMPRSS6 activity in a subject that did not receive the oligonucleotide or pharmaceutical composition or received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control subject).

[0568] In some embodiments of the methods herein, an oligonucleotide (e.g., an RNAi oligonucleotide) provided herein or a pharmaceutical composition comprising the same is administered to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition such that hepcidin production in the subject is increased by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99%, compared to hepcidin production prior to administration of one or more oligonucleotides or pharmaceutical compositions. In some embodiments, the hepcidin production in the subject is increased by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to the hepcidin production in a subject (e.g., a reference or control subject) that did not receive the one or more oligonucleotides or pharmaceutical compositions or received one or more control oligonucleotides, pharmaceutical compositions, or treatments.

[0569] In some embodiments of the methods herein, an oligonucleotide (e.g., RNAi oligonucleotide) provided herein or a pharmaceutical composition comprising the same is administered to a subject having a disease, disorder, or condition associated with hepcidin deficiency or inhibition such that serum iron in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99%, compared to serum iron prior to administration of one or more oligonucleotides or pharmaceutical compositions. In some embodiments, serum iron in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to serum iron in a subject (e.g., a reference or control subject) that did not receive the one or more oligonucleotides or pharmaceutical compositions or received one or more control oligonucleotides, pharmaceutical compositions, or treatments.

[0570] In some embodiments of the methods herein, an oligonucleotide (e.g., RNAi oligonucleotide) provided herein or a pharmaceutical composition comprising the same is administered to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition such that serum iron saturation in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to serum iron saturation prior to administration of one or more oligonucleotides or pharmaceutical compositions. In some embodiments, serum iron saturation in the subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% compared to serum iron saturation in a subject (e.g., a reference or control subject) that did not receive the one or more oligonucleotides or pharmaceutical compositions or received one or more control oligonucleotides, pharmaceutical compositions, or treatments.

[0571] Suitable methods for determining the amount or level of TMPRSS6 expression, TMPRSS6 mRNA, membrane serine protease-2 protein, membrane serine protease-2 activity, or biomarkers (e.g., plasma biomarkers) associated with or affected by TMPRSS6 expression regulation in a subject or in a sample from a subject are known in the art. In addition, the examples presented herein illustrate methods for determining TMPRSS6 expression.

[0572] In some embodiments, the amount or level of TMPRSS6 expression, TMPRSS6 mRNA, membrane-type serpinase-2 protein, membrane-type serpinase-2 activity, or a biomarker associated with or affected by modulation of TMPRSS6 expression, or any combination thereof, is reduced in a cell (e.g., a hepatocyte), a cell population or a group of cells (e.g., an organoid), an organ (e.g., a liver), blood or a component thereof (e.g., plasma), a tissue (e.g., a liver tissue), a sample (e.g., a liver biopsy sample), or any other appropriate biological material obtained or isolated from a subject. In some embodiments, the amount or level of TMPRSS6 expression, TMPRSS6 mRNA, membrane-type serpinase-2 protein, membrane-type serpinase-2 activity, or a biomarker associated with or affected by modulation of TMPRSS6 expression, or any combination thereof, is reduced in more than one type of cell (e.g., hepatocytes and one or more other types of cells), more than one group of cells, more than one organ (e.g., liver and one or more other organs), more than one blood component (e.g., plasma and one or more other blood components), more than one type of tissue (e.g., liver tissue and one or more other types of tissue), or more than one type of sample (e.g., a liver biopsy sample and one or more other types of biopsy samples).

[0573] Due to their high specificity, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) specifically target the mRNA (e.g., TMPRSS6 mRNA) of a target gene in cells and tissues or organs (e.g., in the liver). When preventing a disease, the target gene can be a gene required for the onset or maintenance of the disease, or a gene that has been identified as being associated with a higher risk of developing the disease. When treating a disease, the oligonucleotide can be contacted with a cell, tissue, or organ (e.g., liver) that exhibits or is responsible for mediating the disease. For example, an oligonucleotide (e.g., RNAi oligonucleotide) that is substantially identical to all or part of a wild-type (i.e., natural) or mutant gene associated with a disorder or condition associated with hepcidin deficiency or inhibition can be contacted or introduced into a cell or tissue type of interest, such as a hepatocyte or other liver cell.

[0574] Examples of diseases, disorders or conditions associated with hepcidin deficiency or inhibition include, but are not limited to, hemochromatosis (eg, hereditary hemochromatosis), beta-thalassemia, polycythemia vera, iron-refractory iron-deficiency anemia.

[0575] In some embodiments, the target gene can be a target gene from any mammal, such as a human target.Any target gene can be silenced according to the methods described herein.

[0576] The methods described herein generally involve administering to a subject an effective amount of an oligonucleotide herein (e.g., an RNAi oligonucleotide) that produces or generates an amount of a desired therapeutic outcome. A therapeutically acceptable amount can be an amount that therapeutically treats a disease or condition. The appropriate dosage for any subject will depend on certain factors, including the subject's body size, body surface area, age, composition to be administered, active ingredients in the composition, administration time and route, general health, and other drugs administered simultaneously.

[0577] In some embodiments, any of the compositions herein (e.g., compositions comprising RNAi oligonucleotides described herein) is administered to a subject enterally (e.g., orally, by a gastric feeding tube, by a duodenal feeding tube, via gastrostomy, or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal), topically (e.g., epidermal, inhaled, by eye drops, or through a mucosa), or by direct injection into a target organ (e.g., the subject's liver). Typically, the oligonucleotides herein are administered intravenously or subcutaneously.

[0578] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) or pharmaceutical compositions comprising the oligonucleotides are administered alone or in combination. In some embodiments, the oligonucleotides herein are administered in combination simultaneously, sequentially (in any order) or intermittently. For example, two oligonucleotides can be co-administered simultaneously. Alternatively, one oligonucleotide can be administered, and the second oligonucleotide can be administered after any length of time (e.g., one hour, one day, one week, or one month).

[0579] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats and chickens; and animals such as mice, rats, guinea pigs and hamsters.

[0580] Reagent test kit

[0581] In some embodiments, the disclosure provides a kit comprising an oligonucleotide (e.g., RNAi oligonucleotide) and instructions for use herein. In some embodiments, the kit comprises an oligonucleotide herein, and a package insert comprising instructions for use of the kit and / or any of its components. In some embodiments, the kit comprises an oligonucleotide herein, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art in a suitable container. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container device, in which the oligonucleotide is placed, and in some cases, appropriately aliquoted. In some embodiments providing additional components, the kit comprises an additional container for placing the component. The kit may also include a device for accommodating oligonucleotides, and any other reagents that are strictly limited for commercial sale. Such a container may include an injection-molded or blow-molded plastic container in which the required vial is retained. These containers and / or kits may include labels with instructions for use and / or warnings.

[0582] In some embodiments, the kit comprises an oligonucleotide described herein (e.g., an RNAi oligonucleotide), and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide, and instructions for treating or delaying the progression of a disease, disorder, or condition associated with hepcidin deficiency or inhibition in a subject in need thereof.

[0583] definition

[0584] As used herein, the term "antisense oligonucleotide" encompasses nucleic acid-based molecules having a sequence complementary to all or part of a target mRNA, particularly a seed sequence, thereby being able to form a duplex with the mRNA. Thus, as used herein, the term "antisense oligonucleotide" may be referred to as an "inhibitor based on complementary nucleic acid" or a "guide strand".

[0585] As used herein, "about" or "approximately" when applied to one or more values ​​of interest refers to a value similar to a specified reference value. In certain embodiments, "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a specified reference value in either direction (greater than or less than), unless otherwise specified or apparent from the context (except where the number exceeds 100% of the possible value).

[0586] As used herein, "administering," "administering," and similar terms refer to providing a substance (eg, an oligonucleotide) to a subject in a manner that is pharmacologically useful (eg, treating a disease, disorder, or condition in the subject).

[0587] As used herein, "weaken", "mitigate" and similar terms refer to reducing or effectively preventing. As a non-limiting example, one or more treatments herein can reduce or effectively prevent the onset or progression of iron-refractory iron-deficiency anemia, hemochromatosis or beta-thalassemia in a subject. This weakening can be exemplified by the following facts: for example, one or more aspects (for example, symptoms, tissue characteristics, and cell activity, inflammatory activity or immune activity, etc.) of iron-refractory iron-deficiency anemia, hemochromatosis or beta-thalassemia are reduced, and one or more aspects of hemochromatosis or beta-thalassemia do not have detectable progress (deterioration), or there is no detectable iron-refractory iron-deficiency anemia, hemochromatosis or beta-thalassemia in a subject (and these aspects are originally expected to be detectable).

[0588] As used herein, "complementarity" refers to a structural relationship between two nucleotides (e.g., on two relative nucleic acids or on relative regions of a single nucleic acid chain) that allows the two nucleotides to form base pairs with each other. For example, the purine nucleotides of a nucleic acid complementary to the pyrimidine nucleotides of the relative nucleic acid can be base-paired together by forming hydrogen bonds with each other. In some embodiments, the complementary nucleotides can be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, two nucleic acids may have a region of multiple nucleotides that are complementary to each other to form a complementary region, as described herein.

[0589] As used herein, "deoxyribonucleotide" refers to a nucleotide that has a hydrogen instead of a hydroxyl group at the 2' position of its pentose sugar, as compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide with one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of the sugar, phosphate group, or base, or modifications or substitutions therein.

[0590] As used herein, "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide that is substantially in the form of a duplex. In some embodiments, the complementary base pairing in the duplex region of a double-stranded oligonucleotide is formed between the antiparallel nucleotide sequences of the covalently separated nucleic acid chains. In some embodiments, the complementary base pairing in the duplex region of a double-stranded oligonucleotide is formed between the antiparallel nucleotide sequences of the covalently connected nucleic acid chains. In some embodiments, the complementary base pairing in the duplex region of a double-stranded oligonucleotide is formed by a single nucleic acid chain folded (for example, by a hairpin), to provide a complementary antiparallel nucleotide sequence of base pairing together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that completely form a duplex. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separated nucleic acid chains (for example, at one end or both ends, there is an overhang) that partially forms a duplex. In some embodiments, a double-stranded oligonucleotide comprises partially complementary antiparallel nucleotide sequences, therefore, can have one or more mispairings, which can include internal mispairings or terminal mispairings.

[0591] As used herein, "duplex" with respect to nucleic acids (eg, oligonucleotides) refers to the structure formed by complementary base pairing of two anti-parallel nucleotide sequences.

[0592] As used herein, "excipient" refers to a non-therapeutic agent that can be included in the composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

[0593] As used herein, "hepatocytes" refer to cells of the liver parenchymal tissue. These cells account for about 70%-85% of the liver mass and produce serum albumin, FBN and coagulation factors of the prothrombin group (except factors 3 and 4). Markers of hepatocyte lineage cells include, but are not limited to, transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnf1a) and hepatocyte nuclear factor 4a (Hnf4a). Markers of mature hepatocytes may include, but are not limited to, cytochrome P450 (Cyp3a11), cytochrome P4503A4 (CYP3A4), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb) and OC2-2F8. See, e.g., Huch et al. (2013) Nature 494:247-50.

[0594] As used herein, "hepatotoxic agents" refer to chemical compounds, viruses, or other substances that are toxic to the liver themselves or that can be processed to form metabolites that are toxic to the liver. Hepatotoxic agents may include, but are not limited to, carbon tetrachloride (CCl4), acetaminophen (paracetamol), vinyl chloride, arsenic, chloroform, nonsteroidal anti-inflammatory drugs (such as aspirin and phenylbutazone).

[0595] As used herein, the term "TMPRSS6" refers to transmembrane protease serine 6. The TMPRSS6 gene encodes a membrane serine protease-2 protein that functions with hepcidin in a signaling pathway to regulate iron balance in the body.

[0596] As used herein, a "labile linker" refers to a linker that can be cleaved (eg, by acidic pH). A "fairly stable linker" refers to a linker that cannot be cleaved.

[0597] As used herein, "modified internucleotide linkages" refers to internucleotide linkages having one or more chemical modifications compared to a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, the modified nucleotide is a non-naturally occurring linkage. Typically, the modified internucleotide linkage imparts one or more desired properties to the nucleic acid in which the modified internucleotide linkage is present. For example, the modified internucleotide linkage can improve thermal stability, degradation resistance, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0598] As used herein, "modified nucleotides" refer to nucleotides having one or more chemical modifications compared to corresponding reference nucleotides selected from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, adenine deoxyribonucleotides, guanine deoxyribonucleotides, cytosine deoxyribonucleotides and thymine deoxyribonucleotides. In some embodiments, modified nucleotides are non-naturally occurring nucleotides. In some embodiments, modified nucleotides have one or more chemical modifications in their sugars, nucleobases and / or phosphate groups. In some embodiments, modified nucleotides have one or more chemical moieties conjugated to corresponding reference nucleotides. Typically, modified nucleotides confer one or more desired properties to nucleic acids in which the modified nucleotides are present. For example, modified nucleotides can improve thermal stability, degradation resistance, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc. When referring to modified oligonucleotides, it can be understood to refer to oligonucleotides in which at least one modified nucleotide is included. When referring to a fully modified oligonucleotide, it is understood to refer to an oligonucleotide in which all nucleotides are modified nucleotides. The modified nucleotides do not need to contain the same modification.

[0599] As used herein, a "nicked tetraloop structure" refers to a structure of an RNAi oligonucleotide characterized by separate sense (passenger) and antisense (guide) strands, wherein the sense strand has a complementary region that is complementary to the antisense strand, and wherein at least one strand, typically the sense strand, has a tetraloop configured to stabilize adjacent stem regions formed within the at least one strand. The structure is said to be nicked due to a discontinuity between the backbones of the sense and antisense strands, typically due to a discontinuity between the pentose sugars of adjacent nucleotides of the sense and antisense strands. While the loop may or may not be a tetraloop, a "nicked structure" may generally be considered to have this same definition (e.g., when the sense strand forms a triloop, a tetraloop, or other types of loops as disclosed herein, it may be referred to as a nicked structure).

[0600] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide can be single-stranded (ss) or double-stranded (ds). An oligonucleotide can comprise deoxyribonucleosides, ribonucleosides, or a combination of the two. In some embodiments, a double-stranded oligonucleotide comprising a ribonucleoside is referred to as a "dsRNA". An oligonucleotide may have or may not have a duplex region. As a group of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Dicer substrate interfering RNA (DsiRNA), an antisense oligonucleotide, a short siRNA, or a ss siRNA.

[0601] As used herein, "overhang" refers to a terminal non-base paired nucleotide that is produced by a strand or region extending beyond the end of a complementary strand that forms a duplex with the strand or region. In some embodiments, the overhang comprises one or more unpaired nucleotides extending from the duplex region at the 5' end or 3' end (or its proximal side) of an oligonucleotide. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand of an oligonucleotide.

[0602] As used herein, features may be described as being provided "in," "on," or "at" the 3' or 5' end of a strand. Nucleotide features described as being at the end of a given strand, such as sugar modifications, internucleotide modifications, nucleotide mismatches, and overhangs, are understood to refer to nucleotides of that strand that are proximal to the defined strand end (e.g., at the 3' end of the sense strand).

[0603] As used herein, "phosphate analogs" refer to chemical moieties that simulate the electrostatic properties and / or spatial properties of phosphate groups. In some embodiments, phosphate analogs simulate the electrostatic properties and / or spatial properties of phosphate groups in biological systems. In some embodiments, phosphate analogs are located at the 5' terminal nucleotide of an oligonucleotide, rather than 5'-phosphates that are usually easily removed enzymatically. In some embodiments, 5' phosphate analogs contain phosphatase-resistant connections. Examples of phosphate analogs include, but are not limited to, 5'-phosphonates, such as 5'-methylenephosphonates (5'-MP) and 5'-(E)-vinylphosphonates (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog (referred to as "4'-phosphate analogs") at the 4'-carbon position of the sugar at the 5'-terminal nucleotide. An example of a 4'-phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to a sugar moiety (e.g., at its 4'-carbon) or an analog thereof. See, for example, U.S. Patent Publication No. 2019-0177729. Other modifications have been developed to the 5' end of oligonucleotides (see, e.g., International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) NUCLEICACIDS RES. 43:2993-3011).

[0604] As used herein, "reduced expression" of a gene (e.g., TMPRSS6) refers to a decrease in the amount or level of an RNA transcript (e.g., TMPRSS6 mRNA) or a protein encoded by the gene in a cell, cell population, sample, or subject, and / or a decrease in the amount or level of activity of the gene, compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject). For example, the act of contacting a cell with an oligonucleotide (e.g., an oligonucleotide comprising an antisense strand having a nucleotide sequence complementary to a nucleotide sequence constituting TMPRSS6 mRNA) can result in a decrease in the amount or level of TMPRSS6 mRNA, membrane-type serine protease-2 protein, and / or activity (e.g., via degradation of TMPRSS6 mRNA by an RNAi pathway), compared to cells not treated with the oligonucleotides herein. Similarly, and as used herein, "reduced expression" refers to an act that results in a decrease in the expression of a gene (e.g., TMPRSS6).

[0605] As used herein, "reduction of TMPRSS6 expression" refers to a decrease in the amount or level of TMPRSS6 mRNA, Serpin-2 protein and / or Serpin-2 activity in a cell, cell population, sample or subject compared to an appropriate reference (e.g., a reference cell, cell population, sample or subject).

[0606] As used herein, "complementary region" refers to a nucleotide sequence of a nucleic acid (e.g., an oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence to allow the two nucleotide sequences to hybridize under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.). In some embodiments, the oligonucleotide herein comprises a targeting sequence having a complementary region that is complementary to an mRNA target sequence. The complementary region may have a given length and be identified by a plurality of consecutive nucleotides (e.g., a length of at least 15 consecutive nucleotides), referring to a plurality of nucleotides that are continuously linked together by internucleotide bonding.

[0607] As used herein, "ribonucleotide" refers to a nucleotide having ribose as its pentose, which contains a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of ribose, a phosphate group, or a base, or modifications or substitutions therein.

[0608] As used herein, "RNAi oligonucleotide" refers to (a) a double-stranded oligonucleotide having a sense (passenger) strand and an antisense (guide) strand, wherein the antisense strand or a portion of the antisense strand is used by Argonaute2 (Ago2) endonuclease in the cleavage of a target mRNA (e.g., TMPRSS6 mRNA), or (b) a single-stranded oligonucleotide having a single antisense strand, wherein the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease in the cleavage of a target mRNA (e.g., TMPRSS6 mRNA).

[0609] As used herein, a "strand" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (eg, phosphodiester bonds or phosphorothioate bonds). In some embodiments, a strand has two free ends (eg, a 5' end and a 3' end).

[0610] As used herein, "subject" refers to any mammal, including mice, rabbits, non-human primates (NHPs) and humans. In one embodiment, the subject is a human or NHP. In addition, "individual" or "patient" can be used interchangeably with "subject".

[0611] As used herein, "synthetic" refers to nucleic acids or other molecules that are synthesized artificially (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or that are otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0612] As used herein, "targeting ligand" refers to such a molecule (e.g., a carbohydrate, an amino sugar, cholesterol, a polypeptide or a lipid, such as a GalNAc moiety): it selectively binds to a homologous molecule (e.g., a receptor) of a tissue or cell of interest, and can be conjugated to another substance in order to target the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand can be conjugated to an oligonucleotide in order to target the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Therefore, in some embodiments, when a targeting ligand is conjugated to an oligonucleotide, the delivery of the oligonucleotide to a specific cell is promoted by selective binding to a receptor expressed on the cell surface and endosome internalization of a complex comprising the oligonucleotide, the targeting ligand and the receptor by the cell. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker, which is cleaved after or during cellular internalization so that the oligonucleotide is released from the targeting ligand in the cell.

[0613] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the TMPRSS6 gene (e.g., SEQ ID NO: 853, 854, and 855), including mRNAs that are RNA processing products of primary transcription products. In some embodiments, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-guided cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the TMPRSS6 gene. In some embodiments, the target sequence is located within the protein coding region of the TMPRSS6 gene. In some embodiments, the target sequence is located within the 3'UTR of the TMPRSS6 gene.

[0614] As used herein, "targeting sequence" refers to a nucleotide sequence that is completely or partially complementary to a target sequence.

[0615] As used herein, "loop", "tricycle" or "tetraloop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to each other so that under appropriate hybridization conditions (e.g., in phosphate buffer, in cells), the two antiparallel regions flanking the unpaired region hybridize to form a duplex (referred to as a "stem"). The loop increases the stability of adjacent duplexes formed by hybridization of the flanking sequences of nucleotides. The increase in stability can be detected as a melting temperature (T) of adjacent stem duplexes. m ) increases, the T m higher than the average expected T of adjacent stem duplexes from a set of loops of comparable length consisting of randomly selected nucleotide sequences. mFor example, the loop (e.g., tetraloop or triloop) can confer a T of at least about 50°C, at least about 55°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 65°C, or at least about 75°C to a hairpin comprising a duplex of at least 2 base pairs (bp) in length in 10 mM Na2HPO4. m In some embodiments, the tetraloop can confer a T of at least about 50°C, at least about 55°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 65°C, or at least about 75°C on a hairpin comprising a duplex of at least 2 base pairs (bp) in length in 10 mM NaH2PO4. m. In some embodiments, the loop can stabilize the bp in the adjacent stem duplex by stacking interactions. In addition, the interaction between nucleotides in the loop includes but is not limited to non-Watson-Crick base pairing, stacking interactions, hydrogen bonding and contact interactions (Cheong et al. (1990) Nature 346: 680-82; Heus & Pardi (1991) Science 253: 191-94). In some embodiments, the loop comprises 3 to 6 nucleotides or is composed of 3 to 6 nucleotides, and is generally 4 to 5 nucleotides. In certain embodiments, the loop comprises 3, 4, 5 or 6 nucleotides or is composed of 3, 4, 5 or 6 nucleotides, which may be modified or may not be modified (for example, it may be conjugated with a targeting moiety / targeting ligand or may not be conjugated with a targeting moiety / targeting ligand). In one embodiment, the loop consisting of 3 nucleotides is a tricyclic. In one embodiment, the loop consisting of 4 nucleotides is a tetracyclic. Any nucleotide can be used in a tetraloop, and the standard IUPAC-IUB symbols for such nucleotides can be used as described in Cornish-Bowden (1985) Nucleic Acids Res. 13: 3021-30. For example, the letter "N" can be used to indicate that any base can be in this position, the letter "R" can be used to indicate that A (adenine) or G (guanine) can be in this position, and "B" can be used to indicate that C (cytosine), G (guanine) or T (thymine) can be in this position. Examples of tetraloops include tetraloops of the UNCG family (e.g., UUCG), tetraloops of the GNRA family (e.g., GAAA), and CUUG tetraloops (Woese et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8467-71; Antao et al. (1991) Nucleic Acids Res. 19: 5901-05). Examples of DNA tetraloops include tetraloops of the d(GNNA) family (e.g., d(GTTA)), tetraloops of the d(GNRA) family, tetraloops of the d(GNAB) family, tetraloops of the d(CNNG) family, and tetraloops of the d(TNCG) family (e.g., d(TTCG)). See, e.g., Nakano et al. (2002) Biochem. 41:14281-92; Shinji et al. (2000) Nippon Kagakkai Koen Yokoshu 78:731. In some embodiments, the tetraloop is contained within a gapped tetraloop structure.

[0616] As used herein, "treatment" or "treating" refers to the act of providing medical care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., an oligonucleotide herein) to the subject for improving the health and / or well-being of the subject with respect to an existing condition (e.g., a disease, a disorder), or preventing or reducing the likelihood of a condition occurring. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., a disease, a disorder) experienced by the subject.

[0617] "Pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically suitable for treatment of mammals.

[0618] As used in this article, the symbol Used to describe a connection point.

[0619] List of further embodiments of the invention

[0620] 1. An RNAi oligonucleotide for reducing the expression of transmembrane serine protease 6 (TMPRSS6), the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises a complementary region that is complementary to the TMPRSS6 mRNA target sequence of any one of SEQ ID NOs: 661-852, and wherein the length of the complementary region is at least 15 consecutive nucleotides.

[0621] 2. An RNAi oligonucleotide for reducing the expression of transmembrane serine protease 6 (TMPRSS6), the oligonucleotide comprising a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises a complementary region complementary to the TMPRSS6 mRNA target sequence shown in any one of SEQ ID NOs: 661-852, and wherein the length of the complementary region is at least 15 consecutive nucleotides.

[0622] 3. The RNAi oligonucleotide according to embodiment 1 or 2, wherein the sense strand is 15 to 50 nucleotides in length.

[0623] 4. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the sense strand is 18 to 36 nucleotides in length.

[0624] 5. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the antisense strand has a length of 15 to 30 nucleotides.

[0625] 6. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand and the sense strand form a duplex region of at least 19 nucleotides in length, optionally at least 20 nucleotides in length.

[0626] 7. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand and the sense strand form a duplex region of at least 20 nucleotides in length.

[0627] 8. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the complementary region is at least 18 consecutive nucleotides in length, optionally the complementary region is at least 19 consecutive nucleotides in length.

[0628] 9. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the length of the complementary region is at least 19 consecutive nucleotides.

[0629] 10. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the length of the complementary region is at least 19 consecutive nucleotides.

[0630] 11. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the length of the complementary region is at least 20 consecutive nucleotides.

[0631] 12. A double-stranded RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising:

[0632] (i) an antisense strand having a length of 19-30 nucleotides, wherein the antisense strand comprises a nucleotide sequence comprising a complementary region complementary to a TMPRSS6 mRNA target sequence, wherein the complementary region is selected from the group consisting of SEQ ID NOs: 1-192, and

[0633] (ii) a sense strand of 19-50 nucleotides in length comprising a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric duplex region having an overhang consisting of 1-4 nucleotides at the 3' end of the antisense strand.

[0634] 13. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the 3' end of the sense strand comprises a stem-loop denoted S1-Lp-S2, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3-5 nucleotides in length between S1 and S2.

[0635] 14. The RNAi oligonucleotide of any one of embodiments 1-12, wherein the sense strand comprises a stem-loop denoted S1-Lp-S2 located proximal to the 3' end, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3-5 nucleotides in length between S1 and S2.

[0636] 15. The RNAi oligonucleotide of any one of embodiments 1-12, wherein the sense strand comprises a stem-loop denoted S1-Lp-S2 proximal to the 3' end, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3-5 nucleotides in length between S1 and S2.

[0637] 16. The RNAi oligonucleotide of any one of embodiments 13-15, wherein Lp is a tricyclic or a tetracyclic ring.

[0638] 17. The RNAi oligonucleotide of any one of embodiments 13-16, wherein Lp is a tetraloop.

[0639] 18. The RNAi oligonucleotide of embodiment 17, wherein the tetraloop comprises the sequence 5'-GAAA-3'.

[0640] 19. The RNAi oligonucleotide of any one of embodiments 13-18, wherein the loop is a tetraloop comprising the sequence 5'-GAAA-3'.

[0641] 20. The RNAi oligonucleotide of any one of embodiments 13-19, wherein S1 and S2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides in length.

[0642] 21. The RNAi oligonucleotide of any one of embodiments 13-19, wherein the length of S1 and S2 is each 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides or 10 nucleotides.

[0643] 22. The RNAi oligonucleotide of any one of embodiments 13-21, wherein said S1 and S2 are 1-10 nucleotides in length and have the same length.

[0644] 23. The RNAi oligonucleotide of any one of embodiments 13-22, wherein S1 and S2 are 6 nucleotides in length.

[0645] 24. The RNAi oligonucleotide of any one of embodiments 13-23, wherein the stem-loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 856).

[0646] 25. The RNAi oligonucleotide of any one of embodiments 13-24, wherein a discontinuity is formed between the sense strand and the antisense strand, thereby forming a gapped structure.

[0647] 26. The RNAi oligonucleotide of any one of embodiments 13-25, wherein a discontinuity is formed between the sense strand and the antisense strand, thereby forming a gapped tetraloop structure.

[0648] 27. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the antisense strand comprises a 3' overhang sequence of one or more nucleotides in length.

[0649] 28. The RNAi oligonucleotide of embodiment 27, wherein the overhang comprises a purine nucleotide.

[0650] 29. The RNAi oligonucleotide of any one of embodiments 27-28, wherein the 3' overhang sequence is 2 nucleotides in length.

[0651] 30. The RNAi oligonucleotide of any one of embodiments 27-29, wherein the 3' overhang is selected from the group consisting of AA, GG, AG and GA.

[0652] 31. The RNAi oligonucleotide of embodiment 30, wherein the overhang is GG or AA.

[0653] 32. The RNAi oligonucleotide of any one of embodiments 30-31, wherein the overhang is GG.

[0654] 33. The RNAi oligonucleotide of any one of embodiments 27-29, wherein the 3' overhang is selected from 5'-AA-3', 5'-GG-3', 5'-AG-3' and 5'-GA-3'.

[0655] 34. The RNAi oligonucleotide of embodiment 33, wherein the overhang is 5'-GG-3' or 5'-AA-3'.

[0656] 35. The RNAi oligonucleotide of any one of embodiments 33-34, wherein the overhang is 5'-GG-3'.

[0657] 36. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the oligonucleotide comprises at least one modified nucleotide.

[0658] 37. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the oligonucleotide is fully modified.

[0659] 38. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein all nucleotides of the oligonucleotide are modified nucleotides.

[0660] 39. The RNAi oligonucleotide of any one of embodiments 1-36, wherein the oligonucleotide is partially modified.

[0661] 40. The RNAi oligonucleotide of any one of embodiments 36-39, wherein the modified oligonucleotide comprises a targeting ligand-conjugated nucleotide.

[0662] 41. The RNAi oligonucleotide of any one of embodiments 36-40, wherein the modified nucleotides comprise targeting ligand-conjugated nucleotides.

[0663] 42. The RNAi oligonucleotide of any one of embodiments 36-41, wherein the oligonucleotide comprises a 2'-modification.

[0664] 43. The RNAi oligonucleotide of any one of embodiments 36-42, wherein the modified nucleotide comprises a 2'-modification.

[0665] 44. The RNAi oligonucleotide of any one of embodiments 42-43, wherein the 2'-modification is a modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl and 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid.

[0666] 45. The RNAi oligonucleotide of any one of embodiments 42-44, wherein the 2'-modification is selected from 2'-fluoro (2'-F) and 2'-O-methyl (2'-OMe).

[0667] 46. ​​The RNAi oligonucleotide of any one of embodiments 36-45, wherein the modification is a 2'-modification selected from 2'-fluoro (2'-F) and 2'-O-methyl (2'-OMe).

[0668] 47. The RNAi oligonucleotide of any one of embodiments 36-46, wherein about 10-15%, 10%, 11%, 12%, 13%, 14% or 15% of the nucleotides of the sense strand comprise a 2'-fluoro (2'-F) modification.

[0669] 48. The RNAi oligonucleotide of any one of embodiments 36-47, wherein approximately 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprise a 2'-fluoro (2'-F) modification.

[0670] 49. The RNAi oligonucleotide of any one of embodiments 36-48, wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the oligonucleotide comprise a 2'-fluoro (2'-F) modification.

[0671] 50. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand comprises 36 nucleotides from 5' to 3' positions 1-36, wherein positions 8-11 comprise a 2'-fluoro (2'-F) modification.

[0672] 51. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0673] The sense strand comprises 36 nucleotides, numbered from 5' to 3'; and

[0674] Positions 8-11 of the sense strand all contain 2'-fluoro (2'-F) modifications.

[0675] 52. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0676] The sense strand is 36 nucleotides in length, numbered from 5' to 3'; and

[0677] Positions 8-11 of the sense strand all contain 2'-fluoro (2'-F) modifications.

[0678] 53. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the antisense strand comprises 22 nucleotides from 5' to 3' positions 1-22, and wherein positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro (2'-F) modification.

[0679] 54. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0680] The antisense strand comprises 22 nucleotides, numbered from 5' to 3'; and

[0681] Positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand all contain 2'-fluoro (2'-F) modifications.

[0682] 55. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0683] The antisense strand is 22 nucleotides in length, numbered from 5' to 3'; and

[0684] Positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand all contain 2'-fluoro (2'-F) modifications.

[0685] 56. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand comprises 36 nucleotides from 5' to 3' being positions 1-36, and wherein positions 1-7, 12-27, and 31-36 comprise a 2'-O-methyl modification.

[0686] 57. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0687] The sense strand comprises 36 nucleotides, numbered from 5' to 3'; and

[0688] Positions 1-7, 12-27, and 31-36 of the sense strand all contain 2'-O-methyl modifications.

[0689] 58. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0690] The sense strand is 36 nucleotides in length, numbered from 5' to 3'; and

[0691] Positions 1-7, 12-27, and 31-36 of the sense strand all contain 2'-O-methyl modifications.

[0692] 59. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the antisense strand comprises 22 nucleotides from 5' to 3' being positions 1-22, and wherein positions 1, 6, 8, 9, 11-13 and 15-22 comprise a 2'-O-methyl modification.

[0693] 60. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0694] The antisense strand comprises 22 nucleotides, numbered from 5' to 3'; and

[0695] Positions 1, 6, 8, 9, 11-13 and 15-22 of the antisense strand all contain 2'-O-methyl modifications.

[0696] 61. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0697] The antisense strand is 22 nucleotides in length, numbered from 5' to 3'; and

[0698] Positions 1, 6, 8, 9, 11-13 and 15-22 of the antisense strand all contain 2'-O-methyl modifications.

[0699] 62. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0700] The sense strand comprises 36 nucleotides and the antisense strand comprises 22 nucleotides, the nucleotides of each strand being numbered from 5' to 3';

[0701] Positions 1-7, 12-27, and 31-36 of the sense strand and positions 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand all comprise 2'-O-methyl modifications; and

[0702] Positions 8-11 of the sense strand and positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand all contain 2'-fluoro modifications.

[0703] 63. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the oligonucleotide comprises at least one modified internucleotide linkage.

[0704] 64. The RNAi oligonucleotide of embodiment 63, wherein at least one of the modified internucleotide linkages is a phosphorothioate linkage.

[0705] 65. The RNAi oligonucleotide according to any one of the preceding embodiments, comprising at least one phosphorothioate bond.

[0706] 66. The RNAi oligonucleotide of any of the preceding embodiments, wherein the antisense strand (i) comprises a phosphorothioate bond between positions 1 and 2 and between positions 2 and 3; or (ii) comprises a phosphorothioate bond between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5' to 3'.

[0707] 67. The RNAi oligonucleotide of any of the preceding embodiments, wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises phosphorothioate bonds between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5' to 3'.

[0708] 68. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0709] The antisense strand comprises 22 nucleotides, numbered from 5' to 3'; and

[0710] Phosphorothioate bonds are provided between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22 of the antisense strand.

[0711] 69. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand comprises a phosphorothioate bond between positions 1 and 2, wherein positions are numbered from 5' to 3'.

[0712] 70. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein

[0713] The antisense strand comprises 22 nucleotides;

[0714] The nucleotides of each said strand are numbered from 5' to 3';

[0715] Phosphorothioate bonds are provided between positions 1 and 2 of the sense strand, and between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22 of the antisense strand.

[0716] 71. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog.

[0717] 72. The RNAi oligonucleotide of embodiment 71, wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate or malonylphosphonate, optionally wherein the phosphate analog is a 4'-phosphate analog comprising 4'-oxymethylphosphonate.

[0718] 73. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the 5'-terminal nucleotide of the antisense strand comprises a structure according to Formula 1a (MePhosphonate-4O-mU).

[0719] 74. The RNAi oligonucleotide according to any of the preceding embodiments, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

[0720] 75. The RNAi oligonucleotide of embodiment 74, wherein each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol or a polypeptide.

[0721] 76. The RNAi oligonucleotide of any one of embodiments 13-75, wherein the stem-loop comprises one or more targeting ligands conjugated to one or more nucleotides of the stem-loop.

[0722] 77. The RNAi oligonucleotide of any one of embodiments 74-76, wherein the one or more targeting ligands are conjugated to one or more nucleotides of the loop.

[0723] 78. The RNAi oligonucleotide of any one of embodiments 13-77, wherein the loop (Lp) comprises 4 nucleotides numbered 1-4 from 5' to 3', wherein the nucleotides at positions 2, 3 and 4 each comprise one or more targeting ligands, wherein the targeting ligands are the same or different.

[0724] 79. The RNAi oligonucleotide of any one of embodiments 74-78, wherein each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0725] 80. The RNAi oligonucleotide of embodiment 79, wherein the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0726] 81. The RNAi oligonucleotide of any one of embodiments 79-80, wherein the GalNAc moiety is a monovalent GalNAc moiety.

[0727] 82. The RNAi oligonucleotide of any one of embodiments 13-81, wherein up to 4 nucleotides of Lp of the stem-loop are each conjugated to a monovalent GalNAc moiety.

[0728] 83. The RNAi oligonucleotide of any one of embodiments 13-82, wherein the loop Lp is 4 nucleotides in length, numbered 1-4 from 5' to 3', wherein the nucleotides at positions 2, 3 and 4 of the loop Lp are each conjugated to a monovalent N-acetylgalactosamine (GalNAc) moiety.

[0729] 84. The RNAi oligonucleotide of any one of embodiments 13-82, wherein the loop Lp is 4 nucleotides in length, and wherein the second, third and fourth nucleotides of the loop Lp, counted from 5' to 3', are each conjugated to a monovalent N-acetylgalactosamine (GalNAc) moiety.

[0730] 85. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the complementary region is fully complementary to the mRNA target sequence.

[0731] 86. The RNAi oligonucleotide of any one of embodiments 1-84, wherein the complementary region is partially complementary to the mRNA target sequence.

[0732] 87. The RNAi oligonucleotide of any one of embodiments 1-84 and 86, wherein the region of complementarity comprises no more than 4 mismatches with the mRNA target sequence.

[0733] 88. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the complementary region is fully complementary to the TMPRSS6 mRNA target sequence at nucleotide positions 2-8 of the antisense strand, wherein nucleotide positions are numbered from 5' to 3'.

[0734] 89. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the complementary region is fully complementary to the TMPRSS6 mRNA target sequence at nucleotide positions 2-11 of the antisense strand, wherein nucleotide positions are numbered from 5' to 3'.

[0735] 90. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the TMPRSS6 mRNA target sequence is as shown in SEQ ID NO: 844.

[0736] 91. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 184, and optionally wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 844.

[0737] 92. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 579-580, 585-587, 590, and 595-597.

[0738] 93. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 579-580, 585-587, 590, and 595-597.

[0739] 94. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 600-601, 606-608, 611, and 616-618.

[0740] 95. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 600-601, 606-608, 611, and 616-618.

[0741] 96. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0742] a) SEQ ID NO: 579 and 600, respectively;

[0743] b) SEQ ID NO: 580 and 601, respectively;

[0744] c) SEQ ID NOs: 595 and 616, respectively;

[0745] d) SEQ ID NO: 590 and 611, respectively;

[0746] e) SEQ ID NOs: 596 and 617, respectively;

[0747] f) SEQ ID NOs: 597 and 618, respectively;

[0748] g) SEQ ID NOs: 585 and 606, respectively;

[0749] h) SEQ ID NOs: 586 and 607, respectively; and

[0750] i) are SEQ ID NOs: 587 and 608, respectively.

[0751] 97. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0752] a) SEQ ID NO: 579 and 600, respectively;

[0753] b) SEQ ID NO: 580 and 601, respectively;

[0754] c) SEQ ID NOs: 590 and 611, respectively;

[0755] d) SEQ ID NOs: 597 and 618, respectively; and

[0756] e) are SEQ ID NOs: 586 and 607, respectively.

[0757] 98. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 600, and optionally wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 579.

[0758] 99. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:579, and wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:600.

[0759] 100. The RNAi oligonucleotide of any one of embodiments 1-97, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:580, and wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:601.

[0760] 101. The RNAi oligonucleotide of any one of embodiments 1-97, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:590, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:611.

[0761] 102. The RNAi oligonucleotide of any one of embodiments 1-97, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:597, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:618.

[0762] 103. The RNAi oligonucleotide of any one of embodiments 1-97, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:586, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:607.

[0763] 104. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the antisense strand is 22 nucleotides in length.

[0764] 105. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand is 36 nucleotides in length.

[0765] 106. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0766] a) SEQ ID NO: 621 and 642, respectively;

[0767] b) SEQ ID NO: 622 and 643, respectively;

[0768] c) SEQ ID NOs: 637 and 658, respectively;

[0769] d) SEQ ID NOs: 632 and 653, respectively;

[0770] e) SEQ ID NOs: 638 and 659, respectively;

[0771] f) SEQ ID NOs: 639 and 660, respectively;

[0772] g) SEQ ID NOs: 627 and 648, respectively;

[0773] h) SEQ ID NOs: 628 and 649, respectively; and

[0774] i) are SEQ ID NOs: 629 and 650, respectively.

[0775] 107. The RNAi oligonucleotide of any one of the preceding embodiments, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of:

[0776] a) SEQ ID NO: 621 and 642, respectively;

[0777] b) SEQ ID NO: 622 and 643, respectively;

[0778] c) SEQ ID NOs: 632 and 653, respectively;

[0779] d) SEQ ID NOs: 639 and 660, respectively; and

[0780] e) are SEQ ID NOs: 628 and 649, respectively.

[0781] 108. The RNAi oligonucleotide according to any one of the preceding embodiments, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:621, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:642.

[0782] 109. The RNAi oligonucleotide of any one of embodiments 1-107, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:642, and optionally the sense strand comprises the nucleotide sequence shown in SEQ ID NO:621.

[0783] 110. The RNAi oligonucleotide of any one of embodiments 1-107, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:622, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:643.

[0784] 111. The RNAi oligonucleotide of any one of embodiments 1-107, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:632, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:653.

[0785] 112. The RNAi oligonucleotide of any one of embodiments 1-107, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:639, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:660.

[0786] 113. The RNAi oligonucleotide of any one of embodiments 1-107, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:628, wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:649.

[0787] 114. An RNAi oligonucleotide for reducing the expression of transmembrane serine protease 6 (TMPRSS6), the oligonucleotide comprising a sense strand and an antisense strand forming a duplex region, wherein

[0788] The antisense strand comprises 22 nucleotides and comprises the nucleotide sequence set forth in SEQ ID NO:600, and the sense strand comprises 36 nucleotides and comprises the nucleotide sequence set forth in SEQ ID NO:579, the nucleotides of each strand being numbered from 5' to 3';

[0789] Positions 1-7, 12-27, and 31-36 of the sense strand and positions 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand all comprise a 2'-O-methyl (2'-OMe) modification, and positions 8-11 of the sense strand and positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand all comprise a 2'-fluoro (2'-F) modification;

[0790] providing a phosphorothioate bond between positions 1 and 2 of the sense strand, and between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22 of the antisense strand;

[0791] The sense strand comprises a stem-loop denoted S1-Lp-S2 proximal to the 3' end, S1 is complementary to S2, Lp forms a loop of 4 nucleotides in length, and wherein the second, third and fourth nucleotides counting from 5' to 3' of the loop Lp are each conjugated to a monovalent N-acetylgalactosamine (GalNAc) moiety; and

[0792] The 5'-terminal nucleotide of the antisense strand comprises a structure according to Formula 1a (MePhosphonate-4O-mU):

[0793]

[0794] 115. The RNAi oligonucleotide of embodiment 114, wherein the antisense strand comprises a 3' overhang of one or more purine nucleotides in length, and optionally wherein the 3' overhang is 5'-GG-3'.

[0795] 116. The RNAi oligonucleotide of any one of embodiments 114-115, wherein the nucleotides of the loop Lp conjugated to a monovalent N-acetylgalactosamine (GalNAc) moiety each comprise a structure according to Formula 5a:

[0796]

[0797] 117. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mGs][mG][mU][mG][mC][mU][mA][fC][fU][fC][fU][mG][mG][mU][mA][mU][mU][mU][mC][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:621) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fGs][fAs][fA][fA][mU][fA][mC][mC][fA][mG][mA][mG][fU][mA][mG][mC][mA][mCs][mGs][mG]-3' (SEQ ID NO:642) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein

[0798]

[0799] 118. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein

[0800] The sense strand comprises the sequence of 5'-[mGs][mG][mU][mG][mC][mU][mA][fC][fU][fC][fU][mG][mG][mU][mA][mU][mU][mU][mC][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:621) and all modifications;

[0801] The antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fGs][fAs][fA][fA][mU][fA][mC][mC][fA][mG][mA][mG][fU][mA][mG][mC][mA][mC][mCs][mGs][mG]-3' (SEQ ID NO:642) and all modifications;

[0802] mC, mA, mG, and mU represent 2'-O-methyl (2'-OMe)-modified nucleotides;

[0803] fA, fC, fG, and fU represent 2'-fluoro (2'-F)-modified nucleotides;

[0804] s represents an internucleotide phosphorothioate bond; and

[0805] ademA-GalNAc comprises the structure according to Formula 5a:

[0806]

[0807] 119. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein

[0808] The sense strand comprises the sequence of 5'-[mGs][mG][mU][mG][mC][mU][mA][fC][fU][fC][fU][mG][mG][mU][mA][mU][mU][mU][mC][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:621) and all modifications;

[0809] The antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fGs][fAs][fA][fA][mU][fA][mC][mC][fA][mG][mA][mG][fU][mA][mG][mC][mA][mC][mCs][mGs][mG]-3' (SEQ ID NO:642) and all modifications;

[0810] mC, mA, mG, and mU represent 2'-O-methyl (2'-OMe)-modified nucleotides;

[0811] fA, fC, fG, and fU represent 2'-fluoro (2'-F)-modified nucleotides;

[0812] s represents an internucleotide phosphorothioate bond;

[0813] ademA-GalNAc comprises the structure according to Formula 5a:

[0814] and

[0815] MePhosphonate-4O-mU comprises the structure according to Formula 1a:

[0816]

[0817] 120. An RNAi oligonucleotide for reducing the expression of TMPRSS6, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mGs][mC][mU][mA][mC][mU][mC][fU][fG][fG][fU][mA][mU][mU][mC][mC][mU][mA][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:622) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fUs][fAs][fG][fG][mA][fA][mA][mU][fA][mC][mC][mA][fG][mA][mG][mU][mA][mG][mCs][mGs][mG]-3' (SEQ ID NO:643) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein

[0818]

[0819] 121. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein

[0820] The sense strand comprises the sequence of 5'-[mGs][mC][mU][mA][mC][mU][mC][fU][fG][fG][fU][mA][mU][mU][mU][mC][mC][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:622) and all modifications;

[0821] The antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fUs][fAs][fG][fG][mA][fA][mA][mU][fA][mC][mC][mA][fG][mA][mG][mU][mA][mG][mCs][mGs][mG]-3' (SEQ ID NO: 643) and all modifications

[0822] mC, mA, mG, and mU represent 2'-O-methyl (2'-OMe)-modified nucleotides;

[0823] fA, fC, fG, and fU represent 2'-fluoro (2'-F)-modified nucleotides;

[0824] s represents an internucleotide phosphorothioate bond; and

[0825] ademA-GalNAc comprises the structure according to Formula 5a:

[0826]

[0827] 122. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mCs][mU][mC][mA][mC][mC][mU][fG][fC][fU][fU][mC][mU][mC][mU][mG][mG][mU][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:632) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fAs][fCs][fC][fA][mG][fA][mA][mG][fA][mA][mG][mC][fA][mG][mG][mU][mG][mA][mGs][mGs][mG]-3' (SEQ ID NO:653) and all modifications, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = thiophosphate, and wherein

[0828]

[0829] 123. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein

[0830] The sense strand comprises the sequence of 5'-[mCs][mU][mC][mA][mC][mC][mU][fG][fC][fU][fU][mC][mU][mU][mC][mU][mG][mG][mU][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:632) and all modifications;

[0831] The antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fAs][fCs][fC][fA][mG][fA][mA][mG][fA][mA][mG][mC][fA][mG][mG][mU][mG][mA][mGs][mGs][mG]-3' (SEQ ID NO:653) and all modifications;

[0832] mC, mA, mG, and mU represent 2'-O-methyl (2'-OMe)-modified nucleotides;

[0833] fA, fC, fG, and fU represent 2'-fluoro (2'-F)-modified nucleotides;

[0834] s represents an internucleotide phosphorothioate bond; and

[0835] ademA-GalNAc comprises the structure according to Formula 5a:

[0836]

[0837] 124. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mAs][mG][mU][mG][mU][mG][mA][fA][fA][fG][fA][mC][mA][mU][mA][mG][mC][mU][mG][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO:639) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fCs][fAs][fG][fC][mU][fA][mU][mG][fU][mC][mU][mU][fU][mC][mA][mC][mA][mC][mUs][mGs][mG]-3' (SEQ ID NO:660) and all modifications, wherein mC, mA, mG, mU = 2'-Ome ribonucleoside; fA, fC, fG, fU = 2'-F ribonucleoside; s = thiophosphate, and wherein

[0838]

[0839] 125. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein

[0840] The sense strand comprises the sequence of 5'-[mAs][mG][mU][mG][mU][mG][mA][fA][fA][fG][fA][mC][mA][mU][mA][mG][mC][mU][mG][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO:639) and all modifications;

[0841] The antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fCs][fAs][fG][fC][mU][fA][mU][mG][fU][mC][mU][mU][fU][mC][mA][mC][mA][mC][mUs][mGs][mG]-3' (SEQ ID NO: 660) and all modifications;

[0842] mC, mA, mG, and mU represent 2'-O-methyl (2'-OMe)-modified nucleotides;

[0843] fA, fC, fG, and fU represent 2'-fluoro (2'-F)-modified nucleotides;

[0844] s represents an internucleotide phosphorothioate bond; and

[0845] ademA-GalNAc comprises the structure according to Formula 5a:

[0846]

[0847] 126. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises 5'-[mGs][mG][mG][mU][mG][mC][mA][fC][fU][fA][fU][mG][mG][mC][mU][mU][mG][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO:628) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fUs][fAs][fC][fA][mA][fG][mC][mC][fA][mU][mA][mG][fU][mG][mC][mA][mC][mCs][mGs][mG]-3' (SEQ ID NO:649) and all modifications, wherein mC, mA, mG, mU=2'-OMe ribonucleoside; fA, fC, fG, fU=2'-F ribonucleoside; s=thiophosphate, and wherein

[0848]

[0849] 127. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein

[0850] The sense strand comprises the sequence of 5'-[mGs][mG][mG][mU][mG][mC][mA][fC][fU][fA][fU][mG][mG][mC][mU][mU][mG][mU][mA][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO:628) and all modifications;

[0851] The antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fUs][fAs][fC][fA][mA][fG][mC][mC][fA][mU][mA][mG][fU][mG][mC][mA][mC][mCs][mGs][mG]-3' (SEQ ID NO:649) and all modifications;

[0852] mC, mA, mG, and mU represent 2'-O-methyl (2'-OMe)-modified nucleotides;

[0853] fA, fC, fG, and fU represent 2'-fluoro (2'-F)-modified nucleotides;

[0854] s represents an internucleotide phosphorothioate bond; and

[0855] ademA-GalNAc comprises the structure according to Formula 5a:

[0856]

[0857] 128. The RNAi oligonucleotide of any one of embodiments 116-127, wherein MePhosphonate-4O-mUs comprises a structure according to Formula 1:

[0858]

[0859] 129. The RNAi oligonucleotide of any one of embodiments 116-127, wherein MePhosphonate-4O-mU comprises a structure according to Formula 1a:

[0860]

[0861] 130. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 621 and the antisense strand comprises SEQ ID NO: 642, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig. 10A -The form of the conjugate of the structure shown in B.

[0862] 131. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 621 and the antisense strand comprises SEQ ID NO: 642, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig.14A The conjugate forms of the structures shown.

[0863] 132. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 632 and the antisense strand comprises SEQ ID NO: 653, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig.11A -The form of the conjugate of the structure shown in B.

[0864] 133. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 632 and the antisense strand comprises SEQ ID NO: 653, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig. 14B The conjugate forms of the structures shown.

[0865] 134. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 639 and the antisense strand comprises SEQ ID NO: 660, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig. 12A -The form of the conjugate of the structure shown in B.

[0866] 135. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 639 and the antisense strand comprises SEQ ID NO: 660, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig. 14C The conjugate forms of the structures shown.

[0867] 136. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 628 and the antisense strand comprises SEQ ID NO: 649, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig.13A -The form of the conjugate of the structure shown in B.

[0868] 137. An RNAi oligonucleotide for reducing TMPRSS6 expression, comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 628 and the antisense strand comprises SEQ ID NO: 649, wherein the antisense strand comprises a complementary region to a TMPRSS6 RNA transcript, and wherein the oligonucleotide is Fig.14D The conjugate forms of the structures shown.

[0869] 138. A pharmaceutical composition comprising the RNAi oligonucleotide according to any one of the preceding embodiments and a pharmaceutically acceptable carrier, delivery agent or excipient.

[0870] 139. A method for treating a subject having a disease, disorder or condition associated with hepcidin deficiency or inhibition, the method comprising administering to the subject a therapeutically effective amount of an RNAi oligonucleotide according to any one of embodiments 1-137 or a pharmaceutical composition according to embodiment 138, thereby treating the subject.

[0871] 140. A method according to embodiment 139, wherein after administration of the RNAi oligonucleotide, (i) hepcidin expression is increased; (ii) serum iron levels are reduced; (iii) serum iron saturation is reduced; or (iv) any combination of (i)-(iii).

[0872] 141. A method of delivering an oligonucleotide to a subject, the method comprising administering the pharmaceutical composition of embodiment 138 to the subject.

[0873] 142. A method for reducing TMPRSS6 expression in a cell, a cell population, or a subject, the method comprising the steps of:

[0874] i) contacting the cell or cell population with a RNAi oligonucleotide according to any one of embodiments 1-137 or a pharmaceutical composition according to embodiment 138; or

[0875] ii) administering to the subject the RNAi oligonucleotide according to any one of embodiments 1-137 or the pharmaceutical composition of embodiment 138.

[0876] 143. The method of embodiment 142, wherein reducing TMPRSS6 expression comprises reducing the amount or level of TMPRSS6 mRNA, the amount or level of membrane type serine protease-2 protein, or both.

[0877] 144. A method according to embodiment 142 or 143, wherein reducing TMPRSS6 expression results in (i) increased hepcidin production; (ii) decreased serum iron saturation; (iii) decreased serum iron; or (iv) any combination of (i)-(iii).

[0878] 145. The method of any one of embodiments 142-144, wherein the subject suffers from a disease, disorder, or condition associated with hepcidin deficiency or inhibition.

[0879] 146. The method of any one of embodiments 139-140 and 145, wherein the disease, disorder or condition associated with hepcidin deficiency is hemochromatosis, such as hereditary hemochromatosis.

[0880] 147. The method of any one of embodiments 139-140 and 145, wherein the disease, disorder or condition associated with hepcidin deficiency is β-thalassemia.

[0881] 148. The method of any one of embodiments 139-140 and 145, wherein the disease, disorder, or condition associated with hepcidin inhibition is polycythemia vera.

[0882] 149. The method of any one of embodiments 139-148, wherein the RNAi oligonucleotide or pharmaceutical composition is administered in conjunction with a second composition or therapeutic agent.

[0883] 150. Use of the RNAi oligonucleotide of any one of embodiments 1-137 or the pharmaceutical composition of embodiment 138 in the preparation of a medicament for treating a disease, disorder or condition associated with hepcidin deficiency or inhibition, optionally for treating hemochromatosis such as hereditary hemochromatosis, polycythemia vera or beta-thalassemia.

[0884] 151. The RNAi oligonucleotide according to any one of embodiments 1-137, or the pharmaceutical composition according to embodiment 138, for use as a medicament.

[0885] 152. The RNAi oligonucleotide according to any one of embodiments 1-137, or the pharmaceutical composition according to embodiment 138, for use as a medicament.

[0886] 153. The RNAi oligonucleotide of any one of embodiments 1-137 or the pharmaceutical composition of embodiment 138, which is used or suitable for use in treating a disease, disorder or condition associated with hepcidin deficiency or inhibition, optionally for treating hemochromatosis such as hereditary hemochromatosis, polycythemia vera or beta-thalassemia.

[0887] 154. The RNAi oligonucleotide of any one of embodiments 1-137, or the pharmaceutical composition of embodiment 138, for use in treating hemochromatosis, such as hereditary hemochromatosis.

[0888] 155. The RNAi oligonucleotide of any one of embodiments 1-137, or the pharmaceutical composition of embodiment 138, for use in treating polycythemia vera.

[0889] 156. The RNAi oligonucleotide according to any one of embodiments 1-137, or the pharmaceutical composition according to embodiment 138, for use in treating β-thalassemia.

[0890] 157. The RNAi oligonucleotide according to any one of embodiments 1-137, or the pharmaceutical composition according to embodiment 138, for use in treating hemochromatosis such as hereditary hemochromatosis or β-thalassemia.

[0891] 158. A kit comprising the RNAi oligonucleotide of any one of embodiments 1-137, an optional pharmaceutically acceptable carrier, and a package insert including instructions for administration to a subject suffering from a disease, disorder, or condition associated with hepcidin deficiency or inhibition.

[0892] 159. A kit comprising the RNAi oligonucleotide of any one of embodiments 1-137 or the pharmaceutical composition of embodiment 138 and a package insert comprising instructions for administration to a subject suffering from a disease, disorder or condition associated with hepcidin deficiency or inhibition.

[0893] 160. The use according to embodiment 150, the RNAi oligonucleotide or pharmaceutical composition for use or suitable for use according to any one of embodiments 151-157, or the kit according to any one of embodiments 158-159, wherein the disease, disorder or condition associated with hepcidin deficiency or inhibition is hemochromatosis such as hereditary hemochromatosis, polycythemia vera or beta-thalassemia.

[0894] Example

[0895] Example 1: Preparation of RNAi oligonucleotides

[0896] Oligonucleotide synthesis and purification

[0897] The oligonucleotides described in the previous examples (RNAi oligonucleotides) were chemically synthesized using the methods described herein. In general, except for the use of known phosphoramidite synthesis (see, e.g., Hughes and Ellington (2017) Cold Spring Harb Perspect Biol. 9(1): a023812; Beaucage SL, Caruthers MH Studies on Nucleotide Chemistry V: Deoxynucleoside Phosphoramidites—A New Class of Key Intermediates for Deoxypolynucleotide Synthesis, Tetrahedron In addition to the solid phase oligonucleotide synthesis method described for 19-23-mer siRNA (see, for example, Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441 and Usman et al. (1994)), the solid phase oligonucleotide synthesis method was also used. 987) J. Am. Chem. Soc. 109: 7845-45; see also, U.S. Pat. Nos. 5,804,683, 5,831,071, 5,998,203, 6,008,400, 6,111,086, 6,117,657, 6,353,098, 6,362,323, 6,437,117 and 6,469,158) synthesized RNAi oligonucleotides. The dsRNAi oligonucleotides with a 19-mer core sequence were formatted as constructs with a 25-mer sense strand and a 27-mer antisense strand to allow processing by the RNAi machinery. The 19-mer core sequence is complementary to a region in the TMPRSS6 mRNA.

[0898] Each RNA strand was synthesized and HPLC purified according to standard methods (Integrated DNA Technologies; Coralville, IA). For example, RNA oligonucleotides were synthesized using solid phase phosphoramidite chemistry, deprotected using standard techniques (Damha & Olgivie (1993) Methods Mol. Biol. 20:81-114; Wincott et al. (1995) Nucleic Acids Res. 23:2677-84) and desalted on a NAP-5 column (Amersham Pharmacia Biotech; Piscataway, NJ). Oligos were purified using ion exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm x 25 cm; Amersham Pharmacia Biotech) using a 15 min step linear gradient. The gradient was varied from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris pH 8.5 and buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column and lyophilized.

[0899] The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). The CE capillary had an inner diameter of 100 μm and contained ssDNA 100RGel (Beckman-Coulter). Typically, approximately 0.6 nmol of oligonucleotide was injected into the capillary, run in an electric field of 444 V / cm, and detect...

Claims

1. An RNAi oligonucleotide for reducing the expression of transmembrane serine protease 6 (TMPRSS6), the oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein the antisense strand comprises at least 20 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 600, and wherein the antisense strand comprises a complementary region complementary to a TMPRSS6 mRNA target sequence, and the complementary region is at least 18 consecutive nucleotides.

2. The RNAi oligonucleotide of claim 1, wherein the complementary region is at least 19 consecutive nucleotides.

3. The RNAi oligonucleotide according to any one of claims 1-2, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 844, optionally wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO:

579.

4. The RNAi oligonucleotide according to any one of claims 1 to 3, wherein the antisense strand comprises at least 21 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 600, optionally wherein the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:

600.

5. The RNAi oligonucleotide according to any one of claims 1 to 4, wherein the oligonucleotide comprises at least one modified nucleotide, optionally wherein all nucleotides of the oligonucleotide are modified nucleotides.

6. The RNAi oligonucleotide according to any one of claims 1 to 5, wherein The sense strand comprises 36 nucleotides and the antisense strand comprises 22 nucleotides, the nucleotides of each strand being numbered from 5' to 3'; Positions 1-7, 12-27, and 31-36 of the sense strand and positions 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand all comprise 2'-O-methyl (2'-OMe) modifications; and Positions 8-11 of the sense strand and positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand all contain 2'-fluoro (2'-F) modifications.

7. The RNAi oligonucleotide according to any one of claims 1 to 6, wherein The antisense strand comprises 22 nucleotides; The nucleotides of each said strand are numbered from 5' to 3'; and Phosphorothioate bonds are provided between positions 1 and 2 of the sense strand and between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22 of the antisense strand.

8. The RNAi oligonucleotide according to any one of claims 1 to 7, wherein the 5'-terminal nucleotide of the antisense strand comprises a structure according to Formula 1a (MePhosphonate-4O-mU):

9. The RNAi oligonucleotide according to any one of claims 1 to 8, wherein the sense strand comprises a stem-loop denoted S1-Lp-S2 proximal to the 3' end, wherein S1 is complementary to S2, and wherein Lp forms a loop of 3-5 nucleotides in length between S1 and S2.

10. The RNAi oligonucleotide of claim 9, wherein the loop Lp is 4 nucleotides in length, and wherein the second, third and fourth nucleotides of the loop Lp, counted from 5' to 3', are each conjugated to a monovalent N-acetylgalactosamine (GalNAc) moiety.

11. The RNAi oligonucleotide of any one of claims 1-10, wherein the antisense strand comprises a 3' overhang of one or more purine nucleotides in length, and optionally wherein the 3' overhang is 5'-GG-3'.

12. An RNAi oligonucleotide for reducing TMPRSS6 expression, the oligonucleotide comprising a sense strand and an antisense strand, wherein The sense strand comprises a 5'- [mGs][mG][mU][mG][mC][mU][mA][fC][fU][fC][fU][mG][mG][mU][mA][mU][mU][mU][mC][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mC][mU][mG][mC]-3' (SEQ ID NO:621) and all modifications, and wherein the antisense strand comprises the sequence of 5'-[MePhosphonate-4O-mUs][fGs][fAs][fA][fA][mU][fA][mC][mC][fA][mG][mA][mG][fU][mA][mG][mC][mA][mCs][mGs][mG]-3' (SEQ ID NO:642) and all modifications, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphorothioate, and wherein ademA-GalNAc = 13. A pharmaceutical composition comprising the RNAi oligonucleotide according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier, delivery agent or excipient.

14. The RNAi oligonucleotide according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, for use as a medicament.

15. The RNAi oligonucleotide according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, for use in treating hemochromatosis such as hereditary hemochromatosis or β-thalassemia.

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