NUCLEIC ACIDS FOR INHIBITING EXPRESSION OF TMPRSS6 AND IRON CHELATORS

Abstract

The present invention relates to products and compositions and their uses. In particular the invention relates to nucleic acid products that interfere with the TMPRSS6 gene expression or inhibits its expression in combination with one or more iron chelators and possibly other active agents, as well as therapeutic uses such as for the treatment of hemochromatosis, porphyria and blood disorders such as beta-thalassemia, sickle cell disease and transfusional iron overload or myelodysplastic syndrome, and infections and non-relapse related mortality associated with bone marrow transplantation.

Claims

1. A combination comprising a nucleic acid for inhibiting expression of TMPRSS6 and one or more iron chelators, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, or 442.

2. A combination for use in the treatment of hemochromatosis, porphyria cutanea tarda, blood disorders, such as β-thalassemia or sickle cell disease, congenital dyserythropoietic anemia, marrow failure syndromes, myelodysplasia, transfusional iron overload, a disorder associated with iron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich's Ataxia associated with iron overload, and infections and non-relapse related mortality associated with bone marrow transplantation, preferably of hemochromatosis, the combination comprising a nucleic acid for inhibiting expression of TMPRSS6 and one or more iron chelators, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, or 442.

3. A nucleic acid for use in the treatment of hemochromatosis, porphyria cutanea tarda, blood disorders, such as β-thalassemia or sickle cell disease, congenital dyserythropoietic anemia, marrow failure syndromes, myelodysplasia, transfusional iron overload, a disorder associated with iron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich's Ataxia associated with iron overload, and infections and non-relapse related mortality associated with bone marrow transplantation, preferably of hemochromatosis, wherein the nucleic acid inhibits expression of TMPRSS6, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, or 442, wherein the nucleic acid is administered with one or more iron chelators.

4. A kit comprising a nucleic acid for inhibiting expression of TMPRSS6 and one or more iron chelators, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, or 442.

5. A pharmaceutical composition comprising a nucleic acid for inhibiting expression of TMPRSS6 and one or more iron chelators, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, or 442, wherein the nucleic acid, iron chelator or both are combined with a physiologically acceptable excipient.

6. A pharmaceutical composition for use in the treatment of hemochromatosis, porphyria cutanea tarda, blood disorders, such as β-thalassemia or sickle cell disease, congenital dyserythropoietic anemia, marrow failure syndromes, myelodysplasia, transfusional iron overload, a disorder associated with iron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich's Ataxia associated with iron overload, and infections and non-relapse related mortality associated with bone marrow transplantation, preferably of hemochromatosis, wherein the pharmaceutical composition comprises a nucleic acid for inhibiting expression of TMPRSS6 and one or more iron chelators, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs: 333, 317, 319, 321, 323, 325, 327, 329, 331, 335, 337, 339, 341, 343, 345, 353, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, or 442, wherein the nucleic acid, iron chelator or both are combined with a physiologically acceptable excipient.

7. The combination of claim 1, combination for use of claim 2, nucleic acid for use of claim 3, kit of claim 4, composition of claim 5 or composition for use of claim 6, wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 334, 318, 320, 322, 324, 326, 328, 330, 332, 336, 338, 340, 342, 344, 346, 357, 359. 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, or 443.

8. The combination, combination for use, nucleic acid for use, kit, composition or composition for use according to any preceding claim, wherein the one or more iron chelators are selected from deferoxamine, deferiprone, deferasirox (Exjade) and deferairox (Jadenu), optionally wherein the iron chelator is deferiprone.

9. The combination, combination for use, nucleic acid for use, kit, composition or composition for use according to any preceding claim, wherein one or more nucleotides on the first and/or second strand are modified, to form modified nucleotides.

10. The combination, combination for use, or nucleic acid for use according to claim 9, wherein said first strand comprises a nucleotide sequence of SEQ ID NO:17, and wherein said second strand comprises the nucleotide sequence of SEQ ID NO:18 TABLE-US-00038 SEQ ID 5′aaccagaagaagca 6273646282647284546 NO: 17 gguga 3′ SEQ ID 5′ucaccugcuucuuc 1727354715351718451 NO: 18 ugguu 3′ wherein the specific modifications are depicted by the following numbers 1=2′F-dU, 2=2′F-dA, 3=2′F-dC, 4=2′F-dG, 5=2′-OMe-rU; 6=2′-OMe-rA; 7=2′-OMe-rC; 8=2′-OMe-rG.

11. A combination comprising a nucleic acid for inhibiting expression of TMPRSS6 and deferiprone, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein the first strand comprises the nucleotide sequence of SEQ ID NO:17 and the second strand comprises the nucleotide sequence of SEQ ID NO:18.

12. A combination for use in the treatment of hemochromatosis, porphyria cutanea tarda, blood disorders, such as β-thalassemia or sickle cell disease, congenital dyserythropoietic anemia, marrow failure syndromes, myelodysplasia, transfusional iron overload, a disorder associated with iron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich's Ataxia associated with iron overload, and infections and non-relapse related mortality associated with bone marrow transplantation, preferably hemochromatosis, comprising a nucleic acid for inhibiting expression of TMPRSS6 and deferiprone, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein the first strand comprises the nucleotide sequence of SEQ ID NO:17 and the second strand comprises the nucleotide sequence of SEQ ID NO:18.

13. A nucleic acid for use in the treatment of hemochromatosis, porphyria cutanea tarda, blood disorders, such as β-thalassemia or sickle cell disease, congenital dyserythropoietic anemia, marrow failure syndromes, myelodysplasia, transfusional iron overload, a disorder associated with iron overload, Parkinson's Disease, Alzheimer's Disease or Friedreich's Ataxia associated with iron overload, and infections and non-relapse related mortality associated with bone marrow transplantation, preferably hemochromatosis, wherein the nucleic acid inhibits expression of TMPRSS6, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene, wherein the first strand comprises the nucleotide sequence of SEQ ID NO:17 and the second strand comprises the nucleotide sequence of SEQ ID NO:18, and wherein the nucleic acid is administered with deferiprone.

14. The combination, combination for use, nucleic acid for use, kit, composition or composition for use according to any preceding claim, wherein the nucleic acid and one or more iron chelators are administered simultaneously, separately or sequentially.

15. The combination, combination for use, nucleic acid for use, kit, composition or composition for use according to any preceding claim, wherein the nucleic acid is conjugated to a ligand, optionally at the 5′ end of the second strand, optionally wherein the ligand comprises (i) one or more N-acetyl galactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moieties to the nucleic acid.

16. The combination, combination for use, nucleic acid for use, kit, composition or composition for use according to claim 15, wherein the conjugated nucleic acid has the structure: ##STR00057## ##STR00058## ##STR00059## ##STR00060## wherein Z is a nucleic acid according to any of claims 1 to 14.

17. A combination, combination for use, or nucleic acid for use according to any one of claims 1 to 14, or a conjugated nucleic acid according to claims 15-16, wherein the nucleic acid is stabilised at the 5′ and/or 3′ end of either or both strands, optionally wherein the nucleic acid comprises two phosphorothioate linkages between each of the three terminal 3′ and between each of the three terminal 5′ nucleotides on the first strand, and two phosphorothioate linkages between the three terminal nucleotides of the 3′ end of the second strand, having the structure TABLE-US-00039 5′-3′ TMPRSS6-hcm-9A 6 (ps) 2 (ps) 736462826472845 (ps) 4 (ps) 6 5′-3′ TMPRSS6-hcm-9B 17273547153517184 (ps) 5 (ps) 1 wherein the specific modifications are depicted by the following numbers 1=2′F-dU, 2=2′F-dA, 3=2′F-dC, 4=2′F-dG, 5=2′-OMe-rU; 6=2′-OMe-rA; 7=2′-OMe-rC; 8=2′-OMe-rG (ps)=phosphorothioate linkage.

18. A method of treating a disease or disorder comprising administration of a combination comprising a nucleic acid or conjugated nucleic acid and one or more iron chelators according to any preceding claim to an individual in need of treatment thereof.

Description

[0609] The invention will now be described with reference to the following non-limiting figures and examples in which:

[0610] FIG. 1 shows the results of an RNAi molecule screen for inhibition of TMPRSS6 expression in human Hep3B cells;

[0611] FIG. 2 shows the dose response of TMPRSS6 RNAi molecules in human Hep3B cells;

[0612] FIG. 3 shows the reduction of TMPRSS6 expression in mouse liver tissue by different doses of GalNAc siRNA molecules;

[0613] FIG. 4 shows the duration of TMPRSS6 target gene inhibition by TMPRSS6 siRNA molecules and the induction of HAMP mRNA expression in mice;

[0614] FIG. 5 shows that the inhibition of TMPRSS6 expression by treatment with TMPRSS6 siRNA molecules reduces iron levels in serum for an extended time;

[0615] FIG. 6 shows the reduction of TMRPSS6 expression in 1° mouse hepatocytes by receptor mediated uptake of GalNAc conjugated RNAi molecules;

[0616] FIG. 7 shows the GalNAc conjugated RNAi molecules used in examples 1, 3, 4, 26 to 31 and 34 to 38;

[0617] FIGS. 8a, 8b and 8c show the structure of the GalNAc ligands referred to herein respectively as GN, GN2, and GN3 to which the oligonucleotides were conjugated (see also below this nomenclature in the Examples where TMPRSS6 hcm-9 is conjugated to each of GN, GN2 and GN3);

[0618] FIG. 9 shows the reduction of TMPRSS6 expression by different siRNA modification variants in human Hep3B cells;

[0619] FIG. 10 shows the reduction of TMPRSS6 expression by different GalNAc-siRNA conjugates by receptor mediated uptake;

[0620] FIG. 11 shows the reduction of TMPRSS6 and the induction of HAMP expression by different GalNAc-siRNA conjugates in mice;

[0621] FIG. 12 shows the sequences and modifications of the GalNAc-siRNA molecules of Examples 8, 9 and 10;

[0622] FIG. 13 shows the reduction of TMPRSS6 expression in the liver tissue and the reduction of serum iron levels in mice at different time points after single injection with siRNA conjugates;

[0623] FIG. 14 shows the reduction of TMPRSS6 mRNA levels in liver and the reduction of serum iron levels in mice by different doses of GalNAc conjugated siRNA molecules;

[0624] FIG. 15 shows the effect of different modification patterns on the activity of an siRNA molecule in human Hep3B cells;

[0625] FIG. 16 shows the effect of different modification variants of GalNAc conjugated siRNA molecules on inhibition of TMPRSS6 expression in human Hep3B cells;

[0626] FIG. 17 shows the effect of a modification variant of a GalNAc conjugated siRNAs on inhibition of TMPRSS6 expression in primary mouse hepatocytes by receptor mediated uptake;

[0627] FIG. 18 shows the reduction of TMPRSS6 expression by different siRNA modification variants in human Hep3B cells;

[0628] FIG. 19 shows the influence of inverted A and G nucleotides on RNAi activity in human Hep3B cells;

[0629] FIG. 20 shows the influence of inverted RNA nucleotides on siRNA stability;

[0630] FIG. 21 shows the influence of inverted RNA nucleotides on siRNA activity in human Hep3B cells;

[0631] FIG. 22 shows the influence of inverted RNA nucleotides on RNAi activity in human Hep3B cells;

[0632] FIG. 23 shows the influence of inverted RNA nucleotides on the activity of GalNAc conjugated siRNAs in primary mouse hepatocytes by receptor mediated uptake;

[0633] FIG. 24 shows the effect of phosphorodithioate linkage on stability of GalNAc conjugated siRNA molecules;

[0634] FIG. 25 shows the effect of phosphorodithioate linkage on activity of a GalNAc conjugated siRNA molecule on TMPRSS6 expression in mouse primary hepatocytes;

[0635] FIG. 26 shows the effect of phosphorodithioate linkage on stability of a GalNAc conjugated siRNA molecule;

[0636] FIG. 27 shows the inhibition of TMPRSS6 expression in mouse primary hepatocytes by a GalNAc siRNA conjugate containing phosphorodithioate linkages;

[0637] FIG. 28 shows the reduction of TMPRSS6 expression by different doses of GalNAc siRNAs in an animal model for hereditary hemochromatosis;

[0638] FIG. 29 shows the increase of serum Hepcidin levels by different doses of GalNAc siRNAs in an animal model for hereditary hemochromatosis;

[0639] FIG. 30 shows the reduction of serum iron levels by different doses of GalNAc siRNAs in an animal model for hereditary hemochromatosis;

[0640] FIG. 31 shows the reduction of transferrin saturation by different doses of GalNAc siRNAs in an animal model for hereditary hemochromatosis;

[0641] FIG. 32 shows the increase in Unsaturated Iron Binding Capacity by different doses of GalNAc siRNAs in an animal model for hereditary hemochromatosis;

[0642] FIG. 33 shows the reduction of tissue iron levels by GalNAc siRNAs in an animal model for hereditary hemochromatosis;

[0643] FIG. 34 shows the reduction of human TMPRSS6 mRNA levels in Hep3B cells by liposomal delivery of 43 additional siRNAs;

[0644] FIG. 35 shows the dose response curves of different siRNAs for inhibition of TMPRSS6 expression in Hep3B cells;

[0645] FIG. 36 shows the reduction of TMPRSS6 expression in primary human hepatocytes by receptor mediated uptake of GalNAc siRNA conjugates at different concentrations;

[0646] FIG. 37 shows the increase of haematocrit values in rodent model for β-thalassemia intermedia by treatment with GalNAc siRNA conjugates;

[0647] FIG. 38 shows the reduction in red blood cell distribution widths in rodent model for β-thalassemia intermedia by treatment with GalNAc siRNA conjugates.

[0648] FIG. 39 shows the reduction in proportion of reticulocytes in blood of rodent model for β-thalassemia intermedia by treatment with GalNAc siRNA conjugates;

[0649] FIG. 40 shows the reduction of reactive oxygen species in red blood cells of rodent model for β-thalassemia intermedia by treatment with GalNAc siRNA conjugates.

[0650] FIG. 41 shows GalNAc TMPRSS6 siRNA raises hemoglobin levels in rodent model for β-thalassemia intermedia.

[0651] FIG. 42 shows GalNAc TMPRSS6 reduces splenomegaly in rodent model for β-thalassemia intermedia.

[0652] FIG. 43 shows GalNAc TMPRSS6 improves red blood cell maturation in the bone marrow.

[0653] FIG. 44 shows GalNAc TMPRSS6 reduces ineffective erythropoiesis in the spleen.

[0654] FIG. 45 a and b show dose-response curves of siRNA conjugates against TMPRSS6 in primary human hepatocytes.

[0655] FIG. 46 shows inhibition of TMPRSS6 mRNA expression by siRNA conjugates in primary human hepatocytes.

[0656] FIG. 47 shows sequences and modification pattern of GalNAc siRNA conjugates that were tested for inhibition of TMPRSS6 expression in primary human hepatocytes.

[0657] FIG. 48 shows specific sequences of nucleic acids used in Example 44.

[0658] FIG. 49 shows receptor-mediated uptake in primary mouse hepatocytes by GalNAc siRNA conjugates targeting TMPRSS6 containing different end stabilization chemistries (phosphorothioate, phosphorodithioate, phosphodiester).

[0659] FIG. 50 shows serum stability of GalNAc-siRNA conjugates with phosphorothioates, phosphorodithioates and phosphodiesters in terminal positions and in the GalNAc moiety FIG. 51 shows inhibition of TMPRSS6 gene expression in primary murine hepatocytes 24 h following treatment with TMPRSS6-siRNA carrying vinyl-(E)-phosphonate 2′OMe-Uracil at the 5′-position of the anti-sense strand and two phosphorothioate linkages between the first three nucleotides (X0204), vinyl-(E)-phosphonate 2′OMe-Uracil at the 5′-position of the anti-sense strand and phosphodiester bonds between the first three nucleotides (X0205), (X0139) or tetrameric (X0140)) or a tree like trimeric GalNAc-cluster (X0004) or a non-targeting GalNAc-siRNA (X0028) at indicated concentrations or left untreated (UT).

[0660] FIG. 52 shows serum stability of siRNA-conjugates vs. less stabilized positive control for nuclease degradation.

[0661] FIG. 53 shows reduction of transferrin saturation by TMPRSS6 siRNA treatment in animal model for hereditary hemochromatosis type 1.

[0662] FIG. 54 shows treatment with TMPRSS6 siRNAs enhances the reduction of tissue iron levels by iron chelator.

EXAMPLES

Example 1

[0663] Nucleic acids in accordance with the disclosure were synthesised, using the oligos as set out in the tables below.

[0664] The method of synthesis was as follows, using one of the sequences of the disclosure as an example:

[0665] STS012 (GN-TMPRSS6-hcm-9)

[0666] First strand

[0667] 5′mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA 3′

[0668] Second strand

[0669] 5′[ST23 (ps)]3 long trebler (ps) fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fU 3′

[0670] fN (N=A, C, G, U) denotes 2′Fluoro, 2′ DeoxyNucleosides

[0671] mN (N=A, C, G, U) denotes 2′O Methyl Nucleosides

[0672] (ps) indicates a phosphorothioate linkage

[0673] ST23 is a GalNAc C4 phosphoramidite (structure components as below)

##STR00048##

[0674] Long trebler (STKS)

[0675] A further example is GN2-TMPRSS6-hcm-9 (STS12009L4):

[0676] First strand

[0677] 5′mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA 3′

[0678] Second strand

[0679] 5′[ST23 (ps)]3 ST41 (ps) fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU

[0680] (ps) fU 3′

[0681] fN (N=A, C, G, U) denotes 2′Fluoro, 2′ DeoxyNucleosides

[0682] mN (N=A, C, G, U) denotes 2′O Methyl Nucleosides

[0683] (ps) indicates a phosphorothioate linkage

[0684] ST23 is as above.

[0685] ST41 is as follows (and as described in WO2017/174657):

##STR00049##

[0686] All Oligonucleotides were either obtained from a commercial oligonucleotide manufacturer (Eurogentech, Belgium; Biospring, Germany) or synthesized on an AKTA oligopilot synthesizer using standard phosphoramidite chemistry. Commercially available solid support and 2′O-Methyl RNA phosphoramidtes, 2′Fluoro DNA phosphoramidites (all standard protection) and commercially available long trebler phosphoramidite (Glen research) were used. Synthesis was performed using 0.1 M solutions of the phosphoramidite in dry acetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). All other reagents were commercially available standard reagents.

[0687] Synthesis of PS2 containing oligonucleotides was performed according to the instructions of the manufacturer (Glen Research, AM Biotech). Vinyl-(E)-phosphonate 2′OMe-Uracil phosphoramidite was synthesized and used in oligonucleotide synthesis according to literature published methods (Haraszti et al., Nuc. Acids Res., 45(13), 2017, 7581-7592).

[0688] Conjugation of the GalNAc synthon (ST23) was achieved by coupling of the respective phosphoramidite to the 5′end of the oligochain under standard phosphoramidite coupling conditions. Phosphorothioates were introduced using standard commercially available thiolation reagents (EDITH, Link technologies).

[0689] The single strands were cleaved off the CPG by using Methylamine. Where TBDMS protected RNA nucleosides were used, additional treatment with TEA*3HF was performed to remove the silyl protection. The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 mL, GE Healthcare) on a AKTA Pure HPLC System using a Sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EP Biotech) and lyophilised.

[0690] For annealing, equimolar amounts of the respective single strands were dissolved in water and heated to 80° C. for 5 min. After cooling the resulting Duplex was lyophilised.

[0691] The sequences of the resulting nucleic acids (siNAs) are set out in Table 1.

TABLE-US-00018 TABLE 1 nucleic acid sequences tested for inhibition of TMPRSS6 expression. Nucleic acids were synthesized by Biospring, Frankfurt. Nucleotides modifications are depicted by the following numbers (column 4), 1 = 2′F-dU, 2 = F′-dA, 3 = 2F′-dC, 4 = 2′F-dG, 5 = 2′-OMe-dU; 6 = 2′-OMe-rA; 7 = 2′-OMe-dC; 8 = 2′-OMe-dG. SEQ ID Name - NO: TMPRSS6- . . . Sequence Modifications 1 hc-1A 5′augucuuucacacuggcuu 3′ 6181715172727184715 2 hc-1B 5′aagccagugugaaagacau 3′ 2647364545462646361 3 h-2A 5′auugaguacacgcagacug 3′ 6154645272747282718 4 h-2B 5′cagucugcguguacucaau 3′ 3645354745452717261 5 h-3A 5′aaguugauggugaucccgg 3′ 6281546184546173748 6 h-3B 5′ccgggaucaccaucaacuu 3′ 3748461727361726351 7 hc-4A 5′uucuggaucguccacuggc 3′ 5171846174537271847 8 hc-4B 5′gccaguggacgauccagaa 3′ 4736454827461736462 9 h-5A 5′auucacagaacagaggaac 3′ 6153636462728284627 10 h-5B 5′guuccucuguucugugaau 3′ 4517353545171818261 11 h-6A 5′guagucauggcuguccucu 3′ 8164536184718173535 12 h-6B 5′agaggacagccaugacuac 3′ 2828463647361827163 13 h-7A 5′aguuguaguaaguucccag 3′ 6451816452645173728 14 h-7B 5′cugggaacuuacuacaacu 3′ 3548462715271636271 15 hcmr-8A 5′uuguacccuaggaaauacc 3′ 5181637352846261637 16 hcmr-8B 5′gguauuuccuaggguacaa 3′ 4816151735284816362 17 hcm-9A 5′aaccagaagaagcagguga 3′ 6273646282647284546 18 hcm-9B 5′ucaccugcuucuucugguu 3′ 1727354715351718451 19 hc-10A 5′uaacaacccagcguggaau 3′ 5263627372838184625 20 hc-10B 5′auuccacgcuggguuguua 3′ 2517363835484518152 21 hc-11A 5′guuucucucauccaggccg 3′ 8151717172537284738 22 hc-11B 5′cggccuggaugagagaaac 3′ 3847354825464646263 23 hcm-12A 5′gcaucuucugggcuuuggc 3′ 8361715354847151847 24 hcm-12B 5′gccaaagcccagaagaugc 3′ 4736264737282646183 25 hc-13A 5′ucacacuggaaggugaaug 3′ 5363635482648182618 26 hc-13B 5′cauucaccuuccaguguga 3′ 3615363715372818182 27 hcmr-14A 5′cacagaugugucgaccccg 3′ 7272825454538273738 28 hcmr-14B 5′cggggucgacacaucugug 3′ 3848453827272535454 29 hcmr-15A 5′uguacccuaggaaauacca 3′ 5452737164826252736 30 hcmr-15B 5′ugguauuuccuaggguaca 3′ 1845251537164845272 31 Luc-siRNA-1A 5′ucgaaguauuccgcguacg 3′ 5382645251738381638 32 Luc-siRNA-1B 5′cguacgcggaauacuucga 3′ 3816383856252715382 33 PTEN-A 5′uaaguucuagcuguggugg 3′ 5a6g5u7u6g7u8u8g5g8 34 PTEN-B 5′ccaccacagcuagaacuua 3′ c7a7c6c6g7u6g6a7u5a

TABLE-US-00019 TABLE 2 the start position of each nucleic acid sequence within the TMPRSS6 mRNA sequence Ref NM_001289000.1. SEQ Corresponding ID Start Oligo nucleic acid NO: 253 GGUAUUUCCUAGGGUACAA TMPRSS6-hcmr-8 16 305 CAGUCUGCGUGUACUCAAU TMPRSS6-h-2 4 381 GCCAAAGCCCAGAAGAUGC TMPRSS6-hcm-12 24 426 CUGGGAACUUACUACAACU TMPRSS6-h-7 14 478 UCACCUGCUUCUUCUGGUU TMPRSS6-hcm-9 18 652 AAGCCAGUGUGAAAGACAU TMPRSS6-hc-1 2 682 AUUCCACGCUGGGUUGUUA TMPRSS6-hc-10 20 1234 GCCAGUGGACGAUCCAGAA TMPRSS6-hc-4 8 1318 CCGGGAUCACCAUCAACUU TMPRSS6-h-3 6 1418 GUUCCUCUGUUCUGUGAAU TMPRSS6-h-5 10 1481 CGGCCUGGAUGAGAGAAAC TMPRSS6-hc-11 22 1633 CAUUCACCUUCCAGUGUGA TMPRSS6-hc-13 26 1824 CGGGGUCGACACAUCUGUG TMPRSS6-hcmr-14 28 2018 AGAGGACAGCCAUGACUAC TMPRSS6-h-6 12 252 UGGUAUUUCCUAGGGUACA TMPRRS6-hcmr-15 30

[0692] Screen for Inhibition TMPRSS6 Expression in Human Hep3B Cells.

[0693] Cells were plated at a cell density of 80,000 cells per 6 well dish. The following day cells were transfected with 20 nM nucleic acid (listed in Table 1) and 1 μg/ml AtuFECT (50:50 formulation of cationic lipid Atufect01 and fusogenic lipid DPhyPE.). Two days after transfection cells were lysed and TMPRSS6 mRNA levels were determined by q-RT-PCR using the amplicons in Table 3. TMPRSS6 mRNA levels were normalized to expression levels of the house keeping gene PTEN. Nucleic acids for Luciferase and PTEN were used as non targeting control nucleic acids. Results are shown in FIG. 1.

TABLE-US-00020 TABLE 3 Sequences of TMPRSS6, Actin, PTEN and HAMP amplicon sets that were used to measure mRNA levels of respective genes. SEQ ID NO: hTMPRSS6 (upper) 5′CCGCCAAAGCCCAGAAG 3′ 35 hTMPRSS6 (lower) 5′GGTCCCTCCCCAAAGGAATAG 3′ 36 hTMPRSS6 (probe) 5′CAGCACCCGCCTGGGAACTTACTACAAC 3′ 37 mTMPRSS6 (upper) 5′CGGCACCTACCTTCCACTCTT 3′ 38 mTMPRSS6 (lower) 5′TCGGTGGTGGGCATCCT 3′ 39 mTMPRSS6 (probe) 5′CCGAGATGTTTCCAGCTCCCCTGTTCTA 3′ 40 h-Aktin (upper) 5′GCATGGGTCAGAAGGATTCCTAT 3′ 41 h-Aktin (lower) 5′TGTAGAAGGTGTGGTGCCAGATT 3′ 42 h-Aktin (probe) 5′TCGAGCACGGCATCGTCACCAA 3′ 43 mAktin (upper) 5′GTTTGAGACCTTCAACACCCCA 3′ 44 mAktin (lower) 5′GACCAGAGGCATACAGGGACA 3′ 45 mAktin (probe) 5′CCATGTACGTAGCCATCCAGGCTGTG 3′ 46 PTEN (upper) 5′CACCGCCAAATTTAACTGCAGA 3′ 47 PTEN (lower) 5′AAGGGTTTGATAAGTTCTAGCTGT 3′ 48 PTEN (probe) 5′TGCACAGTATCCTTTTGAAGACCATAACCCA 3′ 49 mHAMP: (upper) 5′CCTGTCTCCTGCTTCTCCTCCT 3′ 50 mHAMP: (lower) 5′AATGTCTGCCCTGCTTTCTTCC 3′ 51 mHAMP: (probe) 5′TGAGCAGCACCACCTATCTCCATCAACA3′ 52

Example 2

[0694] Dose Response of TMPRSS6 Nucleic Acids for Inhibition of TMPRSS6 Expression in Human Hep3B Cells.

[0695] Cells were plated at a cell density of 150,000 cells per 6 well dish. The following day cells were transfected with 1 μg/ml AtuFECT (50:50 formulation of cationic lipid Atufect01 and fusogenic lipid DPhyPE.) and different amounts of TMPRSS6 nucleic acids (30; 10; 3; 1; 0.3; 0.1, 0.003 nM, respectively) as depicted by the X-axis of the graph in FIG. 2. For non targeting control samples, cells were transfected with 30 and 10 nM nucleic acid for Luciferase. Two days after the transfection cells were lysed and mRNA levels were determined by q-RT-PCR. TMPRSS6 mRNA levels were normalized to the expression levels of the house keeping gene PTEN. The highest reduction of TMPRSS6 mRNA expression was observed by TMPRSS6-hcm9 nucleic acid. Transfection with Luciferase nucleic acid did not affect TMPRSS6 mRNA levels. Results are shown in FIG. 2. Sequences of RNAi molecules are depicted in Table 1.

Example 3

[0696] Inhibition of TMPRSS6 Expression in Liver Tissue by Different Doses of GalNAc Nucleic Acids.

[0697] C57/BL6 mice were treated with a single dose of 10, 3 or 1 mg/kg of GalNAc nucleic acid conjugates by subcutaneous administration. Sequence and modifications of respective siRNA conjugates are shown in FIG. 7. The siRNAs are conjugated to GalNAc linker (GN) depicted in FIG. 8a and described therein. Control groups were treated with isotonic saline or with non targeting control conjugate, GN-TTR-hc, respectively. Target gene expression in liver tissue was assessed by qRT PCR three days after subcutaneous injection of the conjugates. Total RNA was isolated from snap frozen tissue samples and qRT-PCR was performed as described previously (Kuhla et al. 2015, Apoptosis Vol 4, 500-11). TaqMan probes that were used are shown in Table 3. Results are shown in FIG. 3.

Example 4

[0698] Duration of Target Gene Inhibition by TMPRSS6 RNAi Molecules.

[0699] Mice were treated with 3 mg/kg GalNAc-TMPRSS6 RNAi molecules by subcutaneous injection. Sequence and modifications of respective siRNA conjugates are shown in FIG. 7. Target mRNA expression was assessed in liver tissue at day 7, 14, 21, 27, 34 or day 41 after treatment (days post injection, dpi). Reduction of TMPRSS6 expression in the liver is observed until day 41 after injection. HAMP (hepcidin) mRNA expression is upregulated in the liver of mice treated with GN-TMPRSS6 RNAi molecule. Results are shown in FIG. 4.

Example 5

[0700] Inhibition of TMPRSS6 Expression by Treatment with TMPRSS6 siRNA Reduces Iron Levels in Blood.

[0701] Iron levels were analyzed in serum 7, 14, 21, 27, 34 and 41 days after mice were treated subcutaneously with 3 mg/kg GalNAc nucleic acid conjugates. Serum iron levels were reduced up to 41 days post injection (dpi 41). Sequence and modifications of respective siRNA conjugates are shown in FIG. 7. Results are shown in FIG. 5.

Example 6

[0702] Inhibition of TMPRSS6 Expression by Receptor Mediated Uptake.

[0703] Primary mouse hepatocytes were plated on collagen coated dishes and incubated with siRNA conjugates diluted in cell culture medium at a concentration of 100 nM to 0.03 nM as indicated. 24 hours after exposing the cells to siRNA conjugates, total RNA was extracted and TMPRSS6 expression was quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to Actin mRNA levels. Dose dependent inhibition of TMPRSS6 expression was observed by both GalNAc-TMPRSS6 siRNA conjugates. GN2-Luc-siRNA1 GalNAc conjugate (GN2-Luc) was used as non targeting control and did not affect TMPRSS6 mRNA expression. Sequence and modifications of respective siRNA conjugates are depicted in FIG. 7. Results are shown in FIG. 6.

Example 7

[0704] Further TMPRSS6 siRNAs were synthesised, in accordance with the method described above. The sequences and modifications are shown in Table 4 below:

[0705] Modification variants of an siRNA targeting TMPRSS6 (Sequence ID 17 and ID18). For each duplex, the first sequence (A strand) is listed on top and the second sequence (B strand) below. All sequences correspond to SEQ ID NO:17 (top) and SEQ ID NO:18 (bottom). Modification codes are listed at the end of the Table 4.

TABLE-US-00021 TABLE 4 Modifications are depicted by numbers shown in the rows at the bottome of the table, and for each duplex the first strand is on top and the second strand is below. Duplex A strand sequence sequence and chemistry ID B strand (5′-3′) (5′-3′) TMP01 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP02 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH02B ucaccugcuucuucugguu 1723314715355754815 TMP03 TMPJH03A aaccagaagaagcagguga 2273282646283248182 TMPJH03B ucaccugcuucuucugguu 5363718351715354815 TMP04 TMPJH04A aaccagaagaagcagguga 2273282646683248182 TMPJH03B ucaccugcuucuucugguu 5363718351715354815 TMP05 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH03B ucaccugcuucuucugguu 5363718351715354815 TMP06 TMPJH05A aaccagaagaagcagguga 2273242242643244542 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP07 TMPJH06A aaccagaagaagcagguga 2277646242643244542 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP08 TMPJH07A aaccagaagaagcagguga 2277686242643244542 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP09 TMPJH08A aaccagaagaagcagguga 2273242246687244542 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP10 TMPJH09A aaccagaagaagcagguga 2273242682687244542 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP11 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP12 TMPJH11A aaccagaagaagcagguga 2273242242643288586 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP13 TMPJH12A aaccagaagaagcagguga 2273242246687284586 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP14 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH13B ucaccugcuucuucugguu 5767354315755758855 TMP15 TMPJH14A aaccagaagaagcagguga 2273282282283284182 TMPJH14B ucaccugcuucuucugguu 5327318315315318415 TMP16 TMPJH15A aaccagaagaagcagguga 2273282282643284182 TMPJH14B ucaccugcuucuucugguu 5327318315315318415 TMP17 TMPJH16A aaccagaagaagcagguga 2237242242247244582 TMPJH16B ucaccugcuucuucugguu 1723314355311358411 TMP18 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP19 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH02B ucaccugcuucuucugguu 1723314715355754815 TMP20 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH03B ucaccugcuucuucugguu 5363718351715354815 TMP21 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH13B ucaccugcuucuucugguu 5767354315755758855 TMP22 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH14B ucaccugcuucuucugguu 5327318315315318415 TMP23 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH16B ucaccugcuucuucugguu 1723314355311358411 TMP24 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP25 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH03B ucaccugcuucuucugguu 5363718351715354815 TMP26 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP27 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH13B ucaccugcuucuucugguu 5767354315755758855 TMP28 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH14B ucaccugcuucuucugguu 5327318315315318415 TMP29 TMPJH02A aaccagaagaagcagguga 2237242282243284142 TMPJH16B ucaccugcuucuucugguu 1723314355311358411 TMP30 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP31 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH02B ucaccugcuucuucugguu 1723314715355754815 TMP32 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH03B ucaccugcuucuucugguu 5363718351715354815 TMP33 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP34 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH14B ucaccugcuucuucugguu 5327318315315318415 TMP35 TMPJH13A aaccagaagaagcagguga 6277646246687284586 TMPJH16B ucaccugcuucuucugguu 1723314355311358411 TMP36 TMPJH10A aaccagaagaagcagguga 2273242242643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP37 TMPJH17A aaccagaagaagcagguga 2273282242643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP38 TMPJH18A aaccagaagaagcagguga 6277682242643288142 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP39 TMPJH19A aaccagaagaagcagguga 6277682242643288586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP40 TMPJH20A aaccagaagaagcagguga 2233282242643284142 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP41 TMPJH21A aaccagaagaagcagguga 2277282242643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP42 TMPJH22A aaccagaagaagcagguga 2273282642643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP43 TMPJH23A aaccagaagaagcagguga 2273282282643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP44 TMPJH24A aaccagaagaagcagguga 2273282246643284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP45 TMPJH25A aaccagaagaagcagguga 2273282242683284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP46 TMPJH26A aaccagaagaagcagguga 2273282242647284586 TMPJH01B ucaccugcuucuucugguu 1727354715351718451 TMP47 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH05B ucaccugcuucuucugguu 5727354315355754455 TMP48 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH27B ucaccugcuucuucugguu 5727754715355754855 TMP49 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH28B ucaccugcuucuucugguu 1723314311351714411 TMP50 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH29B ucaccugcuucuucugguu 5767354315355754455 TMP51 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH30B ucaccugcuucuucugguu 5727754315355754455 TMP52 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH31B ucaccugcuucuucugguu 5727358315355754455 TMP53 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH32B ucaccugcuucuucugguu 5727354315755754455 TMP54 TMPJH01A aaccagaagaagcagguga 6273646282647284546 TMPJH33B ucaccugcuucuucugguu 5727354315355758455 1 = 2′F-dU 2 = 2′F-dA 3 = 2′F-dC 4 = 2′F-dG 5 = 2′OMe-rU 6 = 2′OMe-rA 7 = 2′OMe-rC 8 = 2′OMe-rG

[0706] Different modification variants of one siRNA targeting TMPRSS6 were tested in human Hep3B cells. siRNAs targeting PTEN and Luciferase were used as non-related and non-targeting controls, respectively. All siRNAs were transfected with 1 μg/ml Atufect at 1 nM (0.1 nM when indicated). Total RNA was extracted 48 h after transfection and TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to Actin mRNA levels. Each bar represents mean+/−SD of three technical replicates. The results are shown in FIG. 9.

Example 8

[0707] Modification variants of a GalNAc-conjugated siRNA targeting TMPRSS6 were synthesised and are shown in FIG. 12. For each duplex, the first strand sequence is listed on top and the second strand sequence below. Modifications are depicted as numbers and are as follows: GN indicates conjugation to a GalNAc linker in accordance with FIG. 8a. The sequence and modification of STS12 (GN-TMPRSS6-hcm9) is also depicted in FIG. 7.

Example 9

[0708] Different modification variants of one GalNAc conjugated sequence targeting TMPRSS6 (STS012) reduce TMPRSS6 expression in mouse primary hepatocytes. For receptor-mediated uptake, cells were incubated with 100, 10, 1 and 0.1 nM siRNA conjugate for 24 hours. Total RNA was extracted and TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to Actin mRNA levels. Mean+/−SD of each three technical replicates are shown in FIG. 10.

Example 10

[0709] Different modification variants of one GalNAc-conjugated sequence targeting TMPRSS6 (STS012, GN-TMPRSS6-hcm9) were tested in vivo. 1 mg/kg and 3 mg/kg GalNAc-siRNA conjugate were subcutaneously injected into male C57BL/6JOlaHsd mice. 14 days after treatment, TMPRSS6 (A) and HAMP (B) mRNA levels in the liver were analyzed by Taqman qRT-PCR. Bars represent mean of at least 4 animals+/−SD. Results are shown in FIG. 11.

Example 11

[0710] Two different modification variants of one GalNAc-conjugated sequence targeting TMPRSS6 (STS012) were tested in vivo. 1 mg/kg GalNAc-siRNA conjugates were subcutaneously injected into male C57BL/6JOlaHsd mice. 14 and 28 days after treatment, TMPRSS6 mRNA levels in the liver were analyzed by Taqman qRT-PCR (A). In addition, serum iron levels were analyzed (B). Results are shown in FIG. 13. Box plots represent median of 4 animals. Statistical analysis is based on Kruskal-Wallis test with Dunn's multiple comparison test against PBS group.

[0711] The sequences are as in Example 10.

Example 12

[0712] Two different modification variants of one GalNAc-conjugated sequence targeting TMPRSS6 (STS12009L4, GN2-TMPRSS6-hcm9) were tested in vivo. 1 mg/kg and 0.3 mg/kg GalNAc-siRNA conjugates were subcutaneously injected into male C57BL/6JOlaHsd mice. 14 days after treatment, TMPRSS6 mRNA levels in the liver were analyzed by Taqman qRT-PCR (A). In addition, serum iron levels were analyzed (B). Results are shown in FIG. 14. Box plots represent median of 4 animals. Statistical analysis is based on Kruskal-Wallis test with Dunn's multiple comparison test against PBS group.

[0713] The duplexes used are shown below in Table 5. All sequences correspond to SEQ ID NO:17 and SEQ ID NO:18

TABLE-US-00022 TABLE 5 Modification variants of GalNAc-conjugated sequences targeting TMPRSS6. sequence and chemistry duplex ID (top: first strand, bottom: second strand, both 5′-3′) STS12009L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V2L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-mUmCmAmCfCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU STS12009V8L4 mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mA GN2-mUmCmAmCfCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU GN2-Luc mU(ps)fU(ps)mAfGmUfAmAfAmCfCmUfUmUfUmGfAmG(ps)fA(ps)mC GN2-fGmUfCmUfCmAfAmAfAmGfGmUfUmUfAmCfU(ps)mA(ps)fA mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate GN2 = GalNAc structure according to FIG. 8B

Example 13

[0714] Different modification variants of one siRNA targeting TMPRSS6 were tested in human Hep3B cells. An siRNA targeting Luciferase was used as non-targeting control. All siRNAs were transfected with 1 μg/ml Atufect at 1 nM and 0.1 nM. Total RNA was extracted 48 h after transfection and TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to PTEN mRNA levels. Results are shown in FIG. 15. Each bar represents mean+/−SD of three technical replicates.

[0715] The sequences are shown in Table 6 below. All sequences correspond to SEQ ID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE-US-00023 TABLE 6 Different modification variants of one siRNA targeting TMPRSS6. sequence and chemistry duplex (top: first strand, bottom: second strand, ID both 5′-3′) TMP01 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP66 mAfAmCfCmAmGfAmAmGmAmAmGmCfAmGmGmUmGmA mUmCmAmCmCmUfGmCfUmUmCmUmUmCmUmGmGmUmU TMP69 fAfAfCfCfAmGfAfAfGfAmAfGfCfAmGfGfUfGfA fUmCfAfCfCfUfGfCfUfUfCmUfUmCfUfGfGfUfU TMP79 fAfAmCfCfAmGfAfAfGfAmAfGfCfAmGfGmUmGmA mUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU TMP80 fAfAmCmCfAmGfAfAfGfAmAfGfCfAmGfGmUmGmA mUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU TMP81 fAfAmCfCfAmGfAfAfGfAmAfGmCfAmGfGmUmGmA mUmCfAmCfCmUfGfCfUmUmCmUmUmCmUfGfGmUmU mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate

Example 14

[0716] Modification variants of a GalNAc-conjugated siRNA targeting TMPRSS6 were tested in human Hep3B cells. 150,000 cells were seeded per 6-well. After 24 h, siRNA conjugates were transfected with 1 μg/ml Atufect at 10, 1, 0.1, 0.01, and 0.001 nM (A) or 5, 0.5, 0.05, 0.005 nM (B). A GalNAc-siRNA against Luciferase was used as non-targeting control. Total RNA was extracted 72 h after transfection and TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to PTEN mRNA levels (A) or Actin mRNA levels (B). Results are shown in FIG. 16. Each bar represents mean+/−SD of three technical replicates.

[0717] Sequences are shown in Table 7, below

TABLE-US-00024 TABLE 7 Modification variants of a GalNAc-conjugated siRNA targeting TMPRSS6 sequence and chemistry duplex ID (top: first strand, bottom: second strand, both 5′-3′) STS12009L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V27L4 mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mA GN2-mUmCmAmCmCmUfGfCfUmUmCmUmUmCmUmGmG(ps)mU(ps)mU STS12009V41L4 mA(ps)fA(ps)mCmCmAmGmAmAmGmAmAfGmCfAmGmGmU(ps)mG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate GN2 = GalNAc structure according to FIG. 8B

Example 15

[0718] A modification variant of a GalNAc-conjugated sequence targeting TMPRSS6 (STS12009L4) was tested in mouse primary hepatocytes. For receptor-mediated uptake, cells were incubated with 100, 25, 5, 1, 0.25 and 0.05 nM siRNA conjugate for 24 h. A GalNAc-siRNA targeting an unrelated sequence (GN-TTR) and a non-targeting GalNAc-siRNA (GN-Luc) were used as controls. Total RNA was extracted and TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to PTEN mRNA levels. Results are shown in FIG. 17. Mean+/−SD of each three technical replicates are shown.

[0719] Sequences are shown in Table 7, above.

Example 16

[0720] Different DNA- and LNA-containing variants of one siRNA targeting TMPRSS6 were tested in human Hep3B cells. Therefore, 150,000 cells were seeded per 6-well. After 24 h, siRNAs were transfected with 1 μg/ml Atufect at 0.1 nM siRNA. Total RNA was extracted 48 h after transfection and TMPRSS6 mRNA levels were quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to Actin mRNA levels. Results are shown in FIG. 18. Each bar represents mean+/−SD of three technical replicates.

[0721] Sequences are shown in Table 8, below. All sequences correspond to SEQ ID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE-US-00025 TABLE 8 Different DNA- and LNA-containing variants of one siRNA targeting TMPRSS6. sequence and chemistry duplex (top: first strand, bottom: second ID strand, both 5′-3′) TMP01 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP95 mAfAmCmCmAmGmAmAmGmAmAmGmCfAmGmGmUmGmA fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP99 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA mUmCmAmCmCmUfGmCfUmUmCmUmUmCmUmGmGmUmU TMP112 mA[A]mCmCmAmGmAmAmGmAmAmGmCfAmGmGmUmGmA fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU TMP114 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA mUmCmAmCmCmU{G}mCfUmUmCmUmUmCmUmGmGmUmU TMP115 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA mUmCmAmCmCmUfGmC{U}mUmCmUmUmCmUmGmGmUmU TMP116 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA mUmCmAmCmCmU[G]mCfUmUmCmUmUmCmUmGmGmUmU TMP117 mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA mUmCmAmCmCmUfGmC[U]mUmCmUmUmCmUmGmGmUmU mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA [A], [T], [C], [G] - DNA {A}, {U}, {C}, {G} - LNA (ps) - phosphorothioate

Example 17

[0722] The influence of inverted A and G RNA nucleotides at terminal 3′ positions was analyzed using an siRNA against TMPRSS6. TMP70 contains phosphorothioates at all termini, whereas TMP82 and TMP83 contain ivA (TMP82) and ivG (TMP83) at the 3′-end of the antisense and at the 3′-end of the sense. Both inverted nucleotides are present in addition to the terminal nucleotide of the respective strands and are linked via a phosphorothioate bond. A non-related siRNA (PTEN) and a non-targeting siRNA (Luci) were included as controls. All tested variants show comparable activity under the tested conditions.

[0723] The experiment was conducted in Hep3B cells. Cells were seeded at a density of 150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1 μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extracted and TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR. Results are shown in FIG. 19. Each bar represents mean±SD from three technical replicates.

[0724] The sequences are shown below in Table 9. Sequences correspond to SEQ ID NO:17 (top) or SEQ ID NO:18 (bottom).

TABLE-US-00026 TABLE 9 An siRNA against TMPRSS6 including inverted RNA nucleotides at different positions. duplex sequence and chemistry ID (top: first strand, bottom: second strand, both 5′-3′) TMP70 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU TMP82 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivA fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivA TMP83 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivG fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivG mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA ivA, ivG - inverted RNA (3′-3′) (ps) - phosphorothioate

Example 18

[0725] Different siRNA duplexes containing inverted RNA nucleotides at both 3′-ends were tested for serum stability. TMP84-TMP87 contain inverted RNA in addition to the last nucleotide in the sense strand and instead of the last nucleotide in the antisense strand. TMP88-TMP91 contain inverted RNA in addition to the last nucleotide in the antisense strand and instead of the last nucleotide in the sense strand. All inverted RNA nucleotides substitute for terminally used phosphorothioates. In the design of TMP84-TMP87, ivA and ivG confer higher stability to the tested sequence than ivU and ivC (part A). In the design of TMP88-TMP91, there is no influence of base identity on duplex stability (part B).

[0726] Results are shown in FIG. 20. “UT” indicates untreated samples. “FBS” indicates siRNA duplexes which were incubated at 5 μM final concentration with 50% FBS for 3 d, phenol/chloroform-extracted and precipitated with Ethanol. Samples were analyzed on 20% TBE polyacrylamide gels in native gel electrophoresis.

[0727] Sequences are show in the Table 10, below, and correspond to SEQ ID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE-US-00027 TABLE 10 Different siRNA duplexes containing inverted RNA nucleotides at both 3′-ends. duplex sequence and chemistry ID (top: first strand, bottom: second strand, both 5′-3′) TMP70 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU TMP84 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivA fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP85 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivU fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP86 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivC fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP87 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivG fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG TMP88 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivA TMP89 mA(ps)fA(ps)mCfCmAfGmAfAmGfArnAfGmCfAmGfGmUfGmA ivG fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivU TMP90 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivC TMP91 mA(ps)fA(ps)mCfCmAfGmAfAnnGfAmAfGmCfAmGfGmUfGmA ivG fU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivG mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA ivA, ivU, ivC, ivG - inverted RNA (3′-3′) (ps) - phosphorothioate

Example 19

[0728] The influence of inverted RNA nucleotides at terminal 3′ positions was analyzed using an siRNA against TMPRSS6. TMP70 contains phosphorothioates at all termini, whereas TMP84-TMP87 contain ivG at the 3′-end of the sense strand. The inverted RNA nucleotide is present in addition to the last nucleotide and substitutes for two phosphorothioates. At the antisense 3′-end, ivA (TMP84), ivU (TMP85), ivC (TMP86) and ivG (TMP87) were tested. These inverted RNA nucleotides were added instead of the terminal nucleotide and substitute for phosphorothioates. A non-related siRNA (PTEN) and a non-targeting siRNA (Luci) were included as controls. All tested variants show comparable activity under the tested conditions.

[0729] The experiment was conducted in Hep3B. Cells were seeded at a density of 150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1 μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extracted and TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR. Results are shown in FIG. 21. Each bar represents mean±SD from three technical replicates.

[0730] The sequences are as in Table 10, above.

Example 20

[0731] The influence of inverted RNA nucleotides at terminal 3′ positions was analyzed using an siRNA against TMPRSS6. TMP70 contains phosphorothioates at all termini, whereas TMP88-TMP91 contain ivG at the 3′-end of the antisense strand. The inverted RNA nucleotide is present in addition to the last nucleotide and substitutes for two phosphorothioates. At the sense 3′-end, ivA (TMP88), ivU (TMP89), ivC (TMP90) and ivG (TMP91) were tested. These inverted RNA nucleotides were added instead of the terminal nucleotide and substitute for phosphorothioates. A non-related siRNA (PTEN) and a non-targeting siRNA (Luci) were included as controls. All tested variants show comparable activity under the tested conditions.

[0732] The experiment was conducted in Hep3B. Cells were seeded at a density of 150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1 μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extracted and TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR. Results are shown in FIG. 22. Each bar represents mean±SD from three technical replicates.

[0733] Sequences are as shown in Table 10, above.

Example 21

[0734] The influence of inverted RNA nucleotides at terminal 3′ positions was analyzed using a GalNAc-siRNA conjugate targeting TMPRSS6 in liposomal transfections. STS12009-L4 contains phosphorothioates at all non-conjugated termini, whereas the tested variants contain an inverted RNA nucleotide at the 3′-end of both sense and antisense strand. The inverted RNA is present in addition to the last nucleotide and substitutes for two terminal phosphorothioates (STS12009V10-L4 and -V11-L4) or is used in addition to the terminal phosphorothioates (STS12009V29-L4 and STS12009V30-L4). Inverted A (STS12009V10-L4 and -V29-L4) and inverted G (STS12009V11-L4 and -V30-L4) were used. All tested variants show comparable activity under the tested conditions.

[0735] The experiment was conducted in Hep3B. Cells were seeded at a density of 150,000 cells per 6-well, transfected with 5 nM to 0.0016 nM siRNA and 1 μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extracted and TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR. Results are shown in FIG. 23. Each bar represents mean±SD of three technical replicates.

[0736] Sequences are set out in Table 11, below and correspond to SEQ ID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE-US-00028 TABLE 11 An siRNA sequence including inverted RNA nucleotides at the 3′ ends sequence and chemistry Duplex ID (top: first strand, bottom: second strand, both 5′-3′) STS12009L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V10L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAivA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfUivA STS12009V11L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAivG GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfUivG STS12009V29L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivA STS12009V30L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivG GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivG mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA ivA, ivG - inverted RNA (3′-3′) (ps) - phosphorothioate GN2 = GalNAc structure according to FIG. 8B

Example 22

[0737] Serum stability assay of GalNAc-siRNA conjugates containing one PS2 at individual ends. GalNAc was conjugated to the 5′-end of the sense strand and is internally stabilized by four PS. Phosphorodithioate modifications were placed at the 5′-antisense (STS12009V37L4), 3′-antisense (STS12009V36L4) and 3′-sense (STS12009V34L4) ends. STS12009L4 contains each two terminal PS at 5′-antisense, 3′-antisense and 3′-sense ends, GalNAc is attached to the sense 5′-end and stabilized by four internal PS. 5 μM GalNAc-siRNA conjugates were incubated with 50% FBS for 3 d at 37° C. RNA was extracted and analyzed on 20% TBE polyacrylamide gels. Results are shown in FIG. 24. “UT” indicates untreated samples, “FBS” indicates FBS treatment. “Control” indicates a less stabilized GalNAc-siRNA conjugate of different sequence.

[0738] Sequences are shown in Table 12, below, and correspond to SEQ ID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE-US-00029 TABLE 12 GalNAc-siRNA conjugates containing one PS2 (phosphorodithioate) at individual ends. sequence and chemistry duplex ID (top: first strand, bottom: second strand, both 5′-3′) STS12009L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V34L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU STS12009V36L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG(ps2)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V37L4 mA(ps2)fAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate (ps2) - phosphorodithioate GN2 = GalNAc structure according to FIG. 8B

Example 23

[0739] Activity of GalNAc-siRNA conjugates containing one PS2 at individual ends. GalNAc was conjugated to the 5′-end of the sense strand and is internally stabilized by four PS. Phosphorodithioate modifications were placed at the 5′-antisense (STS12009V37L4), 3′-antisense (STS12009V36L4) and 3′-sense (STS12009V34L4) ends. The experiment was conducted in mouse primary hepatocytes. Cells were seeded at a density of 250,000 cells per 6-well and treated with 100 nM, 10 nM and 1 nM GalNAc-siRNA. Transfections with 10 nM GalNAc-siRNA and 1 μg/ml Atufect served as control. Cells were lysed after 24 h, total RNA was extracted and TMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Results are shown in FIG. 25. Each bar represents mean±SD from three technical replicates.

[0740] Sequences are as set out in Table 12, above.

Example 24

[0741] Serum stability assay of a GalNAc-siRNA conjugate (STS12009V40L4) containing each one PS2 at the second strand 5′-end and at the second strand 3′-end. GalNAc was conjugated to the 5′-end of the second strand and is not stabilized by any internal PS. STS12009L4 contains each two terminal PS at 5′-antisense, 3′-antisense and 3′-sense ends, GalNAc is attached to the sense 5′-end and stabilized by four internal PS. 5 μM GalNAc-siRNA conjugates were incubated with 50% FBS for 3 d at 37° C. RNA was extracted and analyzed on 20% TBE polyacrylamide gels. Results are shown in FIG. 26. “UT” indicates untreated samples, “FBS” indicates FBS treatment. “Control” indicates a less stabilized GalNAc-siRNA conjugate of different sequence.

[0742] Sequences are set out in Table 13, below, and are SEQ ID NO:17 (top) and SEQ ID NO:18 (bottom).

TABLE-US-00030 TABLE 13 GalNAc-siRNA conjugate containing each one PS2 at the second strand 5′-end and at the second strand 3′-end. sequence and chemistry duplex ID (top: first strand, bottom: second strand, both 5′-3′) STS12009L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU STS12009V40L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA GNo-fU(ps2)mCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate (ps2) - phosphorodithioate GN2 - GalNAc, structure according to FIG. 8B GNo - GN2 with phosphodiesters instead of (ps)

Example 25

[0743] Activity of a GalNAc-siRNA conjugate (STS12009V40L4) containing each one PS2 at the sense strand 5′-end and at the sense strand 3′-end. GalNAc was conjugated to the 5′-end of the sense strand and is not stabilized by any internal PS. STS12009L4 contains each two terminal PS at 5′-antisense, 3′-antisense and 3′-sense ends, GalNAc is attached to the sense 5′-end and stabilized by four internal PS. The experiment was conducted in mouse primary hepatocytes. Cells were seeded at a density of 250,000 cells per 6-well and treated with 100 nM, 10 nM and 1 nM GalNAc-siRNA. A GalNAc conjugate of an siRNA against Luciferase (“GalNAc-Luc”) served as control. Cells were lysed after 24 h, total RNA was extracted and TMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Results are shown in FIG. 27. Each bar represents mean±SD from three technical replicates.

[0744] Sequences are as set out in Table 13, above.

Example 26

[0745] Inhibition of TMPRSS6 expression by different doses of GalNAc siRNAs in animal model for hereditary hemochromatosis. HFE female mice (Herrmann et al., J. Mol. Med (Berl), 2004 82, 39-48) were treated subcutaneously with a single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate, respectively. Control groups were treated with PBS or with the non targeting control GN2-Luc siRNA1 (GN2-Luc) by subcutaneous injection. Target gene expression in liver tissue was assessed by qRT PCR three weeks after the injection of the conjugates.

[0746] Group mean and +/−SD. Statistics: Kruskal-Wallis test with uncorrected Dunn. Sequence and modification of siRNA conjugates are depicted in FIG. 7. Results are shown in FIG. 28. P values: ****P<0.001; ***P<0.005; **0.01; *P<0.05.

Example 27

[0747] Increase of serum Hepcidin levels by different doses of GalNAc siRNAs in an animal model for hereditary hemochromatosis. HFE.sup.−/− mice were treated with single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate by subcutaneous injection. Control groups were treated with PBS or with non targeting control conjugate GN2-Luc siRNA 1 (GN2-Luc). Hepcidin levels were determined in serum samples collected three weeks after injection of the conjugates using ELISA kit (Intrinsic Life Science). Group means with SD. Kruskal-Wallis test with uncorrected Dunn's test against control group (GN2-Luc siRNA). Results are shown in FIG. 29. P values: ****P<0.001; ***P<0.005; **0.01; *P<0.05.

Example 28

[0748] Reduction of serum iron levels by different doses of GalNAc siRNAs in animal model for hereditary hemochromatosis. HFE.sup.−/− mice were treated with single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate by subcutaneous administration. Control groups were treated with PBS or with non targeting control conjugate (GN2-Luc siRNA). Serum iron levels were determined three weeks after the treatment. Group means+/−SD. Kruskal-Wallis test with uncorrected Dunn's test against control group (GN2-Luc). Sequences and modifications of siRNA conjugates are depicted in FIG. 7. Results are shown in FIG. 30. P values: ****<0.001; ***P<0.005; **0.01; *P<0.05.

Example 29

[0749] Reduction of transferrin saturation by different doses of GalNAc siRNAs in animal model for hereditary hemochromatosis. HFE.sup.−/− mice were treated with single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate by subcutaneous administration. Control groups were treated with PBS or with non targeting control conjugate GN2-Luc siRNA1 (GN2-Luc). The % transferrin saturation in blood samples was determined three weeks after the treatment. Group means with SD. Kruskal-Wallis test with uncorrected Dunn's test against control group (GN2-Luc). Sequence and modification of siRNA conjugates are depicted in FIG. 7. Results are shown in FIG. 31. P values: ****P<0.001; ***P<0.005; **0.01; *P<0.05.

Example 30

[0750] Increase in Unsaturated Iron Binding Capacity (UIBC) in animal model for hereditary hemochromatosis. HFE.sup.−/− mice were treated with single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate by subcutaneous administration. Control groups were treated with PBS or with non targeting control conjugate GN2-Luc siRNA1 (GN2-LUC). Serum samples were collected three weeks after treatment for determination of UIBC. Group means with SD. Kruskal-Wallis test with uncorrected Dunn's test against control group (GN2-Luc). Results are shown in FIG. 32. P values: ****P<0.001; ***P<0.005; **0.01; *P<0.05.

Example 31

[0751] Reduction of tissue iron levels by GalNAc siRNAs in animal model for hereditary hemochromatosis. HFE.sup.−/− mice were treated with single dose of 1 or 3 mg/kg of GalNAc siRNA conjugate by subcutaneous administration. Control groups were treated with PBS or with non targeting control conjugate GN2-Luc siRNA1 (GN2-Luc). Iron levels in kidney tissue was assessed three weeks after the treatment. Box and Wiskers (Tukey, median values) Kruskal-Wallis test with uncorrected Dunn's test against control group (GN2-Luc ). Results are shown in FIG. 33.

Example 32

[0752] Reduction of TMPRSS6 mRNA expression by different siRNAs in Hep3B cells. 8000 cells per well were plated in 96-well plates. The following day cells were transfected with 20 nM siRNA and 1 μg/ml AtuFECT. Two days after transfection cells were lysed and TMPRSS6 mRNA levels were determined by q-RT-PCR. TMPRSS6 mRNA levels were normalized to expression levels of the house keeping gene ApoB. An siRNAs against Luciferase was used as non targeting control. Average inhibition and standard deviation of triplicate values relative to untreated cells are shown in FIG. 34. The sequences of the siRNAs are shown in Table 14, below.

TABLE-US-00031 TABLE 14 Sequences of siRNAs against TMPRSS6 tested in example 32. fU, fA, fC, fG - 2′F modified deoxynucleotides. mU, mA, mC, mG - 2′O-methyl modified nucleotides. SEQ ID Duplex ID strand ID NO: siRNA sequence TMPRSS6- hcTMP-SR1-A 133 mCfUmGfAmGfGmAfCmGfCmCfCmUfGmGfGmAfGmU SR1 hcTMP-SR1-B 134 fAmCfUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfG TMPRSS6- hcTMP-SR2-A 135 mGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmGfAmG SR2 hcTMP-SR2-B 136 fCmUfCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfC TMPRSS6- hcTMP-SR3-A 137 mUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmGfGmA SR3 hcTMP-SR3-B 138 fUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfA TMPRSS6- hcTMP-SR4-A 139 mGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmG SR4 hcTMP-SR4-B 140 fCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfC TMPRSS6- hcTMP-SR5-A 141 mGfGmUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmG SRS hcTMP-SR5-B 142 fCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfAmCfC TMPRSS6- hcTMP-SR6-A 143 mGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmG SR6 hcTMP-SR6-B 144 fCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfC TMPRSS6- hcTMP-SR8-A 145 mCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmC SR8 hcTMP-SR8-B 146 fGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfG TMPRSS6- hcTMP-SR9-A 147 mAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmGfCmC SR9 hcTMP-SR9-B 148 fGmGfCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfU TMPRSS6- hcTMP-SR10-A 149 mUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmC SR10 hcTMP-SR10-B 150 fGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfA TMPRSS6- hcTMP-SR11-A 151 mGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmG SR11 hcTMP-SR11-B 152 fCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfC TMPRSS6- hcTMP-SR12-A 153 mAfGmUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmC SR12 hcTMP-SR12-B 154 fGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfAmCfU TMPRSS6- hcTMP-SR13-A 155 mAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmA SR13 hcTMP-SR13-B 156 fUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfU TMPRSS6- hcTMP-SR15-A 157 mGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmG SR15 hcTMP-SR15-B 158 fCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfC TMPRSS6- hcTMP-SR16-A 158 mGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmUfGmA SR16 hcTMP-SR16-B 160 fUmCfAmGfCmAfCmCfCmCfGmUfAmCfUmUfCmCfC TMPRSS6- hcTMP-SR17-A 161 mGfGmGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmG SR17 hcTMP-SR17-B 162 fCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfCmCfC TMPRSS6- hcTMP-SR18-A 163 mUfGmGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmU SR18 hcTMP-SR18-B 164 fAmGfCmAfCmCfCmCfGmUfAmCfUmUfCmCfCmCfA TMPRSS6- hcTMP-SR19-A 165 mCfUmGfGmGfGmAfAmGfUmAfCmGfGmGfGmUfGmC SR19 hcTMP-SR19-B 166 fGmCfAmCfCmCfCmGfUmAfCmUfUmCfCmCfCmAfG TMPRSS6- hcTMP-SR21-A 167 mAfGmCfUmGfGmGfGmAfAmGfUmAfCmGfGmGfGmU SR21 hcTMP-SR21-B 168 fAmCfCmCfCmGfUmAfCmUfUmCfCmCfCmAfGmCfU TMPRSS6- hcTMP-SR22-A 169 mUfAmGfCmUfGmGfGmGfAmAfGmUfAmCfGmGfGmG SR22 hcTMP-SR22-B 170 fCmCfCmCfGmUfAmCfUmUfCmCfCmCfAmGfCmUfA TMPRSS6- hcTMP-SR23-A 171 mGfUmAfGmCfUmGfGmGfGmAfAmGfUmAfCmGfGmG SR23 hcTMP-SR23-B 172 fCmCfCmGfUmAfCmUfUmCfCmCfCmAfGmCfUmAfC TMPRSS6- hcTMP-SR24-A 173 mAfGmUfAmGfCmUfGmGfGmGfAmAfGmUfAmCfGmG SR24 hcTMP-SR24-B 174 fCmCfGmUfAmCfUmUfCmCfCmCfAmGfCmUfAmCfU TMPRSS6- hcTMP-SR26-A 175 mGfUmAfGmUfAmGfCmUfGmGfGmGfAmAfGmUfAmC SR26 hcTMP-SR26-B 176 fGmUfAmCfUmUfCmCfCmCfAmGfCmUfAmCfUmAfC TMPRSS6- hcTMP-SR27-A 177 mAfGmUfAmGfUmAfGmCfUmGfGmGfGmAfAmGfUmA SR27 hcTMP-SR27-B 178 fUmAfCmUfUmCfCmCfCmAfGmCfUmAfCmUfAmCfU TMPRSS6- hcTMP-SR28-A 179 mGfAmGfUmAfGmUfAmGfCmUfGmGfGmGfAmAfGmU SR28 hcTMP-SR28-B 180 fAmCfUmUfCmCfCmCfAmGfCmUfAmCfUmAfCmUfC TMPRSS6- hcTMP-SR29-A 181 mCfGmAfGmUfAmGfUmAfGmCfUmGfGmGfGmAfAmG SR29 hcTMP-SR29-B 182 fCmUfUmCfCmCfCmAfGmCfUmAfCmUfAmCfUmCfG TMPRSS6- hcTMP-SR30-A 183 mGfCmGfAmGfUmAfGmUfAmGfCmUfGmGfGmGfAmA SR30 hcTMP-SR30-B 184 fUmUfCmCfCmCfAmGfCmUfAmCfUmAfCmUfCmGfC TMPRSS6- hcTMP-SR31-A 185 mGfGmCfGmAfGmUfAmGfUmAfGmCfUmGfGmGfGmA SR31 hcTMP-SR31-B 186 fUmCfCmCfCmAfGmCfUmAfCmUfAmCfUmCfGmCfC TMPRSS6- hcTMP-SR32-A 187 mGfGmGfCmGfAmGfUmAfGmUfAmGfCmUfGmGfGmG SR32 hcTMP-SR32-B 188 fCmCfCmCfAmGfCmUfAmCfUmAfCmUfCmGfCmCfC TMPRSS6- hcTMP-SR33-A 189 mGfGmGfGmCfGmAfGmUfAmGfUmAfGmCfUmGfGmG SR33 hcTMP-SR33-B 190 fCmCfCmAfGmCfUmAfCmUfAmCfUmCfGmCfCmCfC TMPRSS6- hcTMP-SR34-A 191 mUfGmGfGmGfCmGfAmGfUmAfGmUfAmGfCmUfGmG SR34 hcTMP-SR34-B 192 fCmCfAmGfCmUfAmCfUmAfCmUfCmGfCmCfCmCfA TMPRSS6- hcTMP-SR35-A 193 mUfUmGfGmGfGmCfGmAfGmUfAmGfUmAfGmCfUmG SR35 hcTMP-SR35-B 194 fCmAfGmCfUmAfCmUfAmCfUmCfGmCfCmCfCmAfA TMPRSS6- TMPRSS6-hc-16A 195 mUfAmUfUmCfCmAfAmAfGmGfGmCfAmGfCmUfGmA hc-16 TMPRSS6-hc-16B 196 fUmCfAmGfCmUfGmCfCmCfUmUfUmGfGmAfAmUfA TMPRSS6- TMPRSS6-hc-17A 197 mAfUmCfUmUfCmUfGmGfGmCfUmUfUmGfGmCfGmG hc-17 TMPRSS6-hc-17B 198 fCmCfGmCfCmAfAmAfGmCfCmCfAmGfAmAfGmAfU TMPRSS6- TPRSS6-hc-18A 199 mUfUmUfUmCfUmCfUmUfGmGfAmGfUmCfCmUfCmA hc-18 TMPRSS6-hc-18B 200 fUmGfAmGfGmAfCmUfCmCfAmAfGmAfGmAfAmAfA TMPRSS6- TMPRSS6-hc-19A 201 mGfAmAfUmAfGmAfCmGfGmAfGmCfUmGfGmAfGmU hc-19 TMPRSS6-hc-19B 202 fAmCfUmCfCmAfGmCfUmCfCmGfUmCfUmAfUmUfC TMPRSS6- TMPRSS6-hc-21A 203 mUfAmGfUmAfGmCfUmGfGmGfGmAfAmGfUmAfCmG hc-21 TMPRSS6-hc-21B 204 fCmGfUmAfCmUfUmCfCmCfCmAfGmCfUmAfCmUfA TMPRSS6- TMPRSS6-hc-22A 205 mAfGmAfUmCfCmUfGmGfGmAfGmAfAmGfUmGfGmC hc-22 TMPRSS6-hc-22B 206 fGmCfCmAfCmUfUmCfUmCfCmCfAmGfGmAfUmCfU TMPRSS6- TMPRSS6-hc-23A 207 mCfUmGfUmUfCmUfGmGfAmUfCmGfUmCfCmAfCmU hc-23 TMPRSS6-hc-23B 208 fAmGfUmGfGmAfCmGfAmUfCmCfAmGfAmAfCmAfG TMPRSS6- TMPRSS6-hcmr-24A 209 mCfUmCfAmCfCmUfUmGfAmAfGmGfAmCfAmCfCmU hcmr-24 TMPRSS6-hcmr-24B 210 fAmGfGmUfGmUfCmCfUmUfCmAfAmGfGmUfGmAfG TMPRSS6- TMPRSS6-hcm-25 211 mAfGmUfUmUfCmUfCmUfCmAfUmCfCmAfGmGfCmC hcm-25 TMPRSS6-hcm-25 212 fGmGfCmCfUmGfGmAfUmGfAmGfAmGfAmAfAmCfU TMPRSS6- TMPRSS6-hcr-26A 213 mGfUmAfCmCfCmUfAmGfGmAfAmAfUmAfCmCfAmG hcr-26 TMPRSS6-hcr-26B 214 fCmUfGmGfUmAfUmUfUmCfCmUfAmGfGmGfUmAfC TMPRSS6- TMPRSS6-hc-27A 215 mCfUmGfUmUfGmAfCmUfGmUfGmGfAmCfAmGfCmA hc-27 TMPRSS6-hc-27B 216 fUmGfCmUfGmUfCmCfAmCfAmGfUmCfAmAfCmAfG

Example 33

[0753] Dose-Response of siRNAs Against TMPRSS6 in Hep3B Cells.

[0754] 8000 cells per well were plated in 96-well plates. The following day cells were transfected with siRNA in indicated concentrations (10 nM-0.0 nM) an 1 μg/ml At T Two days after transfection cells were lysed and TMPRSS6 mRNA levels were determined by q-RT-PCR. TMPSS6 mRNA levels were normalized to expression levels of the house keeping gene Apo B. Average inhibition and standard deviation of triplicate values relative to cells treated with 100 nM of anon-targeting Luciferase-control siRNA are shown in FIG. 3. Table 15, blow, shows maximum inhibition, 105 and 95% confidence interval according to dose-response curves shown in FIG. 35.

TABLE-US-00032 TABLE 15 95% kd at Max confidence 20 nM as inhi- IC50 interval shown in Duplex ID bition [nM] [nM] FIG. 34 sd TMPRSS6-hcmr-24 73% 0.8 0.1-6.2  70% 2% TMPRSS-hc-17 82% 2.4 1.1-5.0  79% 5% TMPRSS-SR-27 94% 2.8 0.7-10.8 84% 4% TMPRSS-hcr-26 100%  3.5 1.4-9.0  98% 2% TMPRSS-hc-18 95% 4.6 1.5-14.1 90% 1% TMPRSS-hc-23 83% 4.6 1.9-11.1 70% 7% TMPRSS-hcm-25 87% 4.6 2.1-10.2 85% 3% TMPRSS-SR-21 100%  5.8 0.9-36.9 92% 4% TMPRSS-hc-19 100%  6.1 3.0-12.5 89% 2% TMPRSS-hc-16 100%  7.7 2.6-22.6 91% 3% TMPRSS-hc-22 99% 8.1 2.1-31.1 73% 20%  TMPRSS-SR-16 100%  8.4 2.5-28.2 88% 2% TMPRSS-SR-5 100%  8.4 2.4-29.0 85% 2%

[0755] Table 15: Maximum inhibition, IC50 and 95% confidence interval according to dose response shown in FIG. 35.

Example 34

[0756] Inhibition of TMPSS6 mRNA expression by receptor mediated uptake in 1° human hepatocytes.

[0757] Primary human hepatocytes were plated on collagen coated dishes and incubated with siRNA conjugates diluted in cell culture medium at concentrations of 300 nM to 1 nM as indicated. 24 hours after exposing the cells to siRNA conjugates total RNA was extracted and TMPRSS6 expression was quantified by Taqman qRT-PCR. TMPRSS6 mRNA levels were normalized to Actin mRNA levels and to target mRNA levels of cells treated with non targeting control siRNA conjugate GN2-Luc siRNA 1 (GN2-Luc). Dose dependent inhibition of TMPRSS6 expression was observed by the GalNAc-TMPRSS6 siRNA conjugate.

[0758] Sequences and modifications of the conjugates are depicted in FIG. 7. Results are shown in FIG. 36.

Example 35

[0759] GalNAc TMPRSS6 siRNA Raises Hematocrit Values in Rodent Model for β-Thalassemia Intermedia.

[0760] Hbb.sup.th3/+ mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9 (GN2-TMP), GN2-Luc siRNA 1 (GN2-Luc) or PBS as non targeting control or as vehicle control, respectively. On d 36 whole blood was collected into heparin coated tubes for full blood examination. Hbb.sup.th3/+ mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Blood samples from untreated wild type (WT) mice (C57BL/6) were collected and analysed for comparisons. Scatter dot blot, mean+/−SD; n=3-6. Statistics: unpaired t test with Welch's correction. Results are shown in FIG. 37.

Example 36

[0761] GalNac TMPRSS6 siRNA Reduces Red Blood Cell Distribution Width in Rodent Model for B-Thalassemia Intermedia.

[0762] Hbb.sup.th3/+ mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9 (GN2-TMP), GN2-Luc siRNA1 (GN2-Luc) or PBS as non targeting control or as vehicle control, respectively. On d 36 whole blood was collected into heparin coated tubes for full blood examination. The Hbb.sup.th3/+ mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Blood samples from untreated wild type (WT) mice (C57BL/6) were collected and analysed for comparisons. Scatter dot blot, mean+/−SD; n=3-6. Statistics: unpaired t test with Welch's correction. Results are shown in FIG. 38.

Examples 37

[0763] GalNAc TMPRSS6 siRNA reduces the proportion of reticulocytes in rodent model for β-thalassemia intermedia.

[0764] Hbb.sup.th3/+ mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6 hcm9 (GN2-TMP), GN2-Luc siRNA 1 (GN2-Luc) or PBS as non targeting control or as vehicle control, respectively. On d 36 whole blood was collected into heparin coated tubes for full blood examination. The Hbb.sup.th3/+ mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Blood samples from untreated wild type (WT) mice (C57BL/6) were collected and analysed for comparisons. Scatter dot blot, mean+/−SD; n=3-6. Statistics: unpaired t test with Welch's correction. Results are shown in FIG. 39.

Examples 38

[0765] GalNAc TMPRSS6 siRNA reduces the amount of reactive oxygen species (ROS) in rodent model for β-thalassemia intermedia. Hbb.sup.th3/+ mice (Th3/+; Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6 hcm9 (GN2-TMP) or GN2-Luc 1 siRNA (GN2-Luc) as non targeting control. On d 36 whole blood was collected into heparin coated tubes for full blood examination. ROS measurements were performed 5 min. after addition of 2′7′ dichloro-fluorescein as indicator (Siwaponanan et al, 2017 Blood, 129, 3087-3099). Blood samples from GN2-TMPRSS6 hcm9 treated mice were measured twice. The Hbb.sup.th3/+ mice (Th3/+) were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Blood samples from untreated wild type (WT) mice (C57BL/6) were collected and analysed for comparisons. Scatter dot blot, mean+/−SD; n=4-6. Statistic: unpaired t test with Welch's correction. Results are shown in FIG. 40.

Example 39

[0766] GalNAc TMPRSS6 siRNA raises hemoglobin levels in rodent model for β-thalassemia intermedia. Hbb.sup.th3/+ mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9 (GN2-TMP), GN2-Luc-siRNA 1 (GN2-Luc) or PBS as non targeting control or as vehicle control, respectively. On d 36 whole blood was collected into heparin coated tubes for full blood examination. Hbb.sup.th3/+ mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Blood samples from untreated wild type (wt) mice (C57BL/6) were collected and analysed for comparisons. Scatter dot blot, mean+/−SD; n=5-7. Statistics: Welch's t-tests uncorrected for multiple comparison. Results are shown in FIG. 41. siRNA conjugates are depicted in FIG. 7.

Example 40

[0767] GalNAc TMPRSS6 reduces splenomegaly in rodent model for β-thalassemia intermedia. Hbbt.sup.th3/+ mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9 (GN2-TMP), GN2-Luc-siRNA 1 (GN2-Luc) or PBS as non targeting control or as vehicle control, respectively. On d 39 spleen weights were assessed. Hbb.sup.th3/+ mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Spleen weights from wild type (wt) mice (C57BL/6) treated with PBS were assessed for comparisons. Scatter dot blot, mean+/−SD; n=5-7. Statistics: Welch's t-tests uncorrected for multiple comparison. Results are shown in FIG. 42. siRNA conjugates are depicted in FIG. 7.

Example 41

[0768] GalNAc TMPRSS6 improves red blood cell maturation in the bone marrow. Hbb.sup.th3/+ mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6-hcm9 (GN2-TMP), GN2-Luc-siRNA 1 (GN2-Luc) or PBS as non targeting control or as vehicle control, on d39 cells were collected from the bone marrow and analyzed by FACS analysis. Viable erythroid cells were separated into distinct populations based on CD71, Ter119 and CD44 staining. Hbb.sup.th3/+ mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Erythroid cells from the bone marrow of wild type (wt) mice (C57BL/6) treated with PBS were assessed for comparisons. Bar graph, mean+/−SD; n=5-7. Statistics: Welch's t-tests uncorrected for multiple comparison. Results are shown in FIG. 43. siRNA conjugates are depicted in FIG. 7.

Example 42

[0769] GalNAc TMPRSS6 reduces ineffective erythropoiesis in the spleen. Hbb.sup.th3/+ mice (Yang et al. 1995, PNAS Vol. 92, 11608-11612) were treated on d1 and on d15 subcutaneously with 3 mg/kg GN2-TMPRSS6 hcm9 (GN2-TMP), GN2-Luc siRNA 1 (GN2-Luc) or PBS as non targeting control or as vehicle control, respectively. On d 39 cells were collected from the spleen and analyzed by FACS analysis. Viable erythroid cells were separated into distinct populations based on CD71, Ter119 and CD44 staining. Hbb.sup.th3/+ mice were obtained from Jackson Laboratory (Bar Harbor, Me.) and maintained on a C57BL/6 background. Erythroid cells from the from wild type (wt) mice (C57BL/6) treated with PBS were assessed for comparisons. Bar graph, mean+/−SD; n=5-7. Statistics: Welch's t-tests uncorrected for multiple comparison. Results are shown in FIG. 44. siRNA conjugates are depicted in FIG. 7.

Example 43

[0770] Reduction of TMPRSS6 Expression in Human Hepatocytes by GalNAc siRNA Conjugates.

[0771] 30,000 cpw human primary hepatocytes were seeded in collagen-coated 96-well plates. Cell were treated with indicated amounts of siRNA conjugates immediately after plating. Cells were lysed 24 hours post treatment and TMPRSS6 mRNA expression analyzed by TaqMan qRT-PCR. Triplicate values of TMPRSS6 normalized to ApoB (housekeeper) and to mean of untreated cells are shown.

[0772] Results are shown in FIG. 45a, 45b and FIG. 46, and sequences are shown in FIG. 47. The GN3 linker described is shown in FIG. 8C.

Example 44

[0773] Serum stability assay of GalNAc-siRNA conjugates with phosphorothioates, phosphorodithioates and phosphodiesters in terminal positions and in the GalNAc moiety. GalNAc was conjugated to the 5′-end of the sense strand and is internally stabilized by four PS (STS12009L4) or not, then phosphodiester linkages are used instead (STS12009V54L50—V57L50). Phosphorodithioate modifications were placed at all terminal positions of the duplex except of the first strand 5′-end (−V54L50), at the 3′-ends only (−V55L50, −V57L50) or at the 3′-end of the second strand only (−V56L50). In certain designs, phosphodiesters were used in terminal positions of the siRNA duplex (−V56L50, −V57L50). 5 μM GalNAc-siRNA conjugates were incubated with 50% FBS for 3 d at 37° C. RNA was extracted and analyzed on 20% TBE polyacrylamide gels. “UT” indicates untreated samples, “FBS” indicates FBS treatment. “Control” indicates a less stabilized GalNAc-siRNA conjugate of different sequence.

[0774] GalNAc conjugates of an siRNA targeting TMPRSS6 containing different end stabilization chemistries (phosphorothioate, phosphorodithioate, phosphodiester) were tested by receptor-mediated uptake in primary mouse hepatocytes. GalNAc was conjugated to the 5′-end of the second strand and is internally stabilized by four PS (STS12009L4) or not, then phosphodiester linkages are used instead (STS12009V54L50—V57L50). Phosphorodithioate modifications were placed at all terminal positions of the duplex except of the first strand 5′-end (−V54L50), at the 3′-ends only (−V55L50, −V57L50) or at the 3′-end of the second strand only (−V56L50). In certain designs, phosphodiesters were used in terminal positions of the siRNA duplex (−V56L50, −V57L50). The experiment was conducted in mouse primary hepatocytes. Cells were seeded at a density of 20,000 cells per 96-well and treated with 125 nM to 0.2 nM GalNAc-siRNA. Cells were lysed after 24 h, total RNA was extracted and TMPRSS6 and PTEN mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean±SD of three technical replicates.

[0775] Results and relevant sequences are shown in FIGS. 48-50.

Example 45

[0776] Reduction of TMPRSS6 Expression in Primary Murine Hepatocytes by GalNAc siRNA Conjugates with 2′-O-Methyl-Uridine or 5′-(E)-Vinylphosphonate-2′-O-Methyl-Uridine Replacing the 2′-O-Methyl-Adenin at the 5′ Position of the First Strand.

[0777] Murine primary hepatocytes were seeded into collagen pre-coated 96 well plates (Thermo Fisher Scientific, #A1142803) at a cell density of 30,000 cells per well and treated with siRNA-conjugates at concentrations ranging from 100 nM to 0.1 nM. 24 h post treatment cells were lysed and RNA extracted with InviTrap® RNA Cell HTS 96 Kit/C24×96 preps (Stratec #7061300400) according to the manufactures protocol. Transcripts levels of TMPRSS6 and housekeeping mRNA (Ptenll) were quantified by TaqMan analysis.

[0778] siRNA Conjugates:

TABLE-US-00033 first strand/ siRNA second duplex strand sequence & modification STS12009L4 TMPRSS6- mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG (X0027) hcm9-A fG mU (ps) fG (ps) mA TMPRSS6- GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm9-BL4 fG (ps) mU (ps) fU STS12209V4L4 TMPRSS6- vinylphosphonate-mU (ps) fA (ps) mC fC mA fG mA fA mG fA (X0204) hcm209AV4 mA fG mC fA mG fG mU (ps) fG (ps) mA TMPRSS6- GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4 fG (ps) mU (ps) fA STS12209V5L4 TMPRSS6- vinylphosphonate-mU fA mC fC mA fG mA fA mG fA mA fG (x0205) hcm209-AV5 mC fA mG fG mU (ps) fG (ps) mA TMPRSS6- GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4 fG (ps) mU (ps) fA STS12209L4 TMPRSS6- mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG (x0207) hcm209A fG mU (ps) fG (ps) mA TMPRSS6- GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4 fG (ps) mU (ps) fA STS12209V1L4 TMPRSS6- mU fA mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (x0208) hcm9-AV1 (ps) fG (ps) mA TMPRSS6- GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG hcm209-BL4 fG (ps) mU (ps) fA STS18001 STS18001A mU(ps)fC(ps)mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA(ps) (X0028) fC(ps)mG STS18001BL4 GN2 fCmGfUmAfCmGfCmGfGmAfAmUfAmCfUmUfC (ps) mG (ps) fA Legend: mN 2′-O-methyl ribonucleotide (e.g. mU - 2′-O-methyl Uracil) fN 2′-fluoro ribonucleotide (e.g. fC - 2′-fluoro Cytidin) (ps) phosphorothioate vinylphosphonate vinyl-(E)-phosphonate GN2 strcuture according to FIG. 8B

[0779] TaqMan Primer and Probes

TABLE-US-00034 PTEN-2 CACCGCCAAATTTAACTGCAGA PTEN-2 AAGGGTTTGATAAGTTCTAGCTGT PTEN-2 FAM-TGCACAGTATCCTTTTGAAGACCATAAC CCA-TAMRA hTMSS6:379U17 CCGCCAAAGCCCAGAAG hTMSS6:475L21 GGTCCCTCCCCAAAGGAATAG hTMSS6:416U28FL FAM-CAGCACCCGCCTGGGAACTTACTACAAC- BHQ1

[0780] Legend:

[0781] FAM—6-carboxyfluorescein (fluorescent dye)

[0782] TAMRA—tetramethylrhodamine (quencher)

[0783] BHQ1—black hole quencher 1 (quencher)

[0784] In Vitro Dose Response

[0785] Target gene expression in primary murine hepatocytes 24 h following treatment with TMPRSS6-siRNA carrying vinyl-(E)-phosphonate 2′OMe-Uracil at the 5′-position of the anti-sense strand and two phosphorothioate linkages between the first three nucleotides (STS12209V4L4), vinyl-(E)-phosphonate 2′OMe-Uracil at the 5′-position of the anti-sense strand and phosphodiester bonds between the first three nucleotides (STS12209V5L4), carrying 2′-O-methyl-Uracil and two phosphorothioate linkages between the first three nucleotides at the 5′-position (STS12209L4) or carrying 2′-O-methyl-Uracil and two phosphodiester linkages between the first three nucleotides at the 5′-position (STS12209V1L4) or ) or (STS12009L4) as reference or a non-targeting GalNAc-siRNA (STS18001) at indicated concentrations or left untreated (UT).

[0786] Results are shown in FIG. 51.

[0787] Serum Stability

[0788] Serum stability of siRNA conjugates incubated for 4 hours (4 h) or 3 days (3 d) or left untreated (0 h) in 50% FCS at 37° C. Following RNA was extracted by phenol/chlorophorm/isoamyl alcohol extraction. Degradation was visualized by TBE-Polyacrylamid-gel-electrophoresis and staining RNA with SybrGold.

[0789] Results are shown in FIG. 52: Serum stability of siRNA-conjugates vs. less stabilized positive control for nuclease degradation.

Example 46

[0790] Reduction of transferrin saturation by TMPRSS6 siRNA treatment in animal model for hereditary hemochromatosis type 1. Three months old C57BL6/J congenic female HFE knock-out mice (−/−) and three months old female C57BL6/J wild type mice (+/+) were used in this study (Herrmann et al., Mol. Med, 2004. 82 (1): p1-3). HFE −/− mice (n=8) were treated on Day 1 (1×) or on Day 1 and on Day 22 (2×) subcutaneously with either PBS (PBS) or 3 mg/kg TMPRSS6 siRNA conjugate (TMP) or 3 mg/kg non-targeting siRNA conjugate (Luc). In addition, cohort 7 to 10 received Deferiprone supplied in drinking water at a concentration of 0.5 mg/ml. Cohorts 1 and 2 were terminated on Day 1 (Predose) all other cohorts on Day 43 (6 weeks after start of the treatment) to collect serum and tissue samples for analyses. Serum iron levels (SFBC) and unsaturated iron binding capacity UIBC were assessed by calorimetric methods. Transferrin saturation (Tf Sat) was calculated using the following formula: TfSat=(SFBC/TIBC)×100. Transferrin saturation is elevated in 3 months old HFE −/− mice at start of the treatment phase (cohort 2, Predose). Treatment with TMPRSS6 siRNA conjugates alone or in combination with Deferiprone reduced transferrin saturation in this rodent model for hereditary hemochromatosis and iron overload.

[0791] Results are shown in FIG. 53.

[0792] siRNA Conjugates:

TABLE-US-00035 siRNA conjugate Single strands Sequence and chemistry TMP TMP-A Strand 5′mA (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC SEQ ID NO: 217 fA mG fG mU (ps) fG (ps) mA 3′ TMP-B Strand 5′GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU SEQ ID NO: 218 mG fG (ps) mU (ps) fU 3′ Luc Luc-A 5′mU(ps)fC(ps)mGfAmAfGmUfAmUfUmCfCmGfCmGfUm A (ps) fC(ps) mG 3′ Luc-B 5′GN2 fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mU fC (ps) mG (ps) fA 3′ Legend: fN (N = A, C, G, U) denotes 2′Fluoro, 2′DeoxyNucleosides mN (N = A, C, G, U) denotes 2′O Methyl Nucleosides (ps) indicates a phosphorothioate linkage GN2 GalNAc structure as in FIG. 8B TMP-A is identical to SEQ ID NO: 111 TMP-B is identical to SEQ ID NO: 112, except evidently that GN has been replaced by GN2 TMP-A base sequence is identical to SEQ ID NO: 333 TMP-B base sequence is identical to SEQ ID NO: 334 TMP-A modification pattern is identical to SEQ ID NO: 17 TMP-B modification pattern is identical to SEQ ID NO: 18 Luc-A is identical to SEQ ID NO: 109 Luc-B is identical to SEQ ID NO: 110 Luc-A base sequence is identical to SEQ ID NO: 347 Luc-B base sequence is identical to SEQ ID NO: 348 Luc-A modification pattern is identical to SEQ ID NO: 31 Luc-B modification pattern is identical to SEQ ID NO: 32

Example 47

[0793] Treatment with TMPRSS6 siRNAs enhances the reduction of tissue iron levels by iron chelator. 3 months old HFE −/− (rodent model for hereditary hemochromatosis and iron overload) or wild type mice were used for this study and treated as described in Example 46. Liver tissue samples were collected to assess liver iron concentrations before start of the treatment (predose) and at the end of the study at Day 43 (6 weeks after start of treatments). Liver tissue samples were dried and iron concentrations from dry weights were determined by acid extraction method. At the start of the study liver iron levels were already elevated in HFE −/− compared to wild type control mice (Predose). Treatment of HFE −/− mice with TMPRSS6 siRNA conjugates in combination with Deferiprone enhanced the reduction of tissue iron levels by the iron chelator. In other words, the combined siRNA/iron chelator treatment led to a much greater reduction of iron content in the liver than was achieved by the treatment with the siRNA or the chelator alone (compare columns 5 and 9 or 6 and 10). This is surprising because the treatment with the siRNA alone (compare column 3 with columns 5 and 6) or with the iron chelator alone (column 7) has little to no effect on the liver iron content. The liver iron content is further reduced by administration of multiple doses of the siRNA (compare columns 9 and 10).

[0794] siRNA conjugates are as described in Example 46 and results are shown in FIG. 54.

TABLE-US-00036 Summary SEQUENCE TABLE SEQ ID NO Name Sequence (5′-3′) 1 TMPRSS6-hc-1A 6181715172727184715 2 TMPRSS6-hc-1B 2647364545462646361 3 TMPRSS6-h-2A 6154645272747282718 4 TMPRSS6-h-2B 3645354745452717261 5 TMPRSS6-h-3A 6281546184546173748 6 TMPRSS6-h-3B 3748461727361726351 7 TMPRSS6-hc-4A 5171846174537271847 8 TMPRSS6-hc-4B 4736454827461736462 9 TMPRSS6-h-5A 6153636462728284627 10 TMPRSS6-h-5B 4517353545171818261 11 TMPRSS6-h-6A 8164536184718173535 12 TMPRSS6-h-6B 2828463647361827163 13 TMPRSS6-h-7A 6451816452645173728 14 TMPRSS6-h-7B 3548462715271636271 15 TMPRSS6-hcmr-8A 5181637352846261637 16 TMPRSS6-hcmr-8B 4816151735284816362 17 TMPRSS6-hcm-9A 6273646282647284546 18 TMPRSS6-hcm-9B 1727354715351718451 19 TMPRSS6-hc-10A 5263627372838184625 20 TMPRSS6-hc-10B 2517363835484518152 21 TMPRSS6-hc-11A 8151717172537284738 22 TMPRSS6-hc-11B 3847354825464646263 23 TMPRSS6-hcm-12A 8361715354847151847 24 TMPRSS6-hcm-12B 4736264737282646183 25 TMPRSS6-hc-13A 5363635482648182618 26 TMPRSS6-hc-13B 3615363715372818182 27 TMPRSS6-hcmr-14A 7272825454538273738 28 TMPRSS6-hcmr-14B 3848453827272535454 29 TMPRSS6-hcmr-15A 5452737164826252736 30 TMPRSS6-hcmr-15B 1845251537164845272 31 TMPRSS6-Luc-siRNA-1A 5382645251738381638 32 TMPRSS6-Luc-siRNA-1B 3816383856252715382 33 TMPRSS6-PTEN-A 5a6g5u7u6g7u8u8g5g8 34 TMPRSS6-PTEN-B c7a7c6c6g7u6g6a7u5a 35 hTMPRSS6 (upper) ccgccaaagcccagaag 36 hTMPRSS6 (lower) ggTcccTccccaaaggaaTag 37 hTMPRSS6 (probe) cagcacccgccTgggaacTTacTacaac 38 mTMPRSS6 (upper) cggcaccTaccTTccacTcTT 39 mTMPRSS6 (lower) TcggTggTgggcaTccT 40 mTMPRSS6 (probe) ccgagaTgTTTccagcTccccTgTTcTa 41 h-Aktin (upper) gcaTgggTcagaaggaTTccTaT 42 h-Aktin (lower) TgTagaaggTgTggTgccagaTT 43 h-Aktin (probe) TcgagcacggcaTcgTcaccaa 44 mAktin (upper) gTTTgagaccTTcaacacccca 45 mAktin (lower) gaccagaggcaTacagggaca 46 mAktin (probe) ccaTgTacgTagccaTccaggcTgTg 47 PTEN (upper) caccgccaaaTTTaacTgcaga 48 PTEN (lower) aagggTTTgaTaagTTcTagcTgT 49 PTEN (probe) TgcacagTaTccTTTTgaagaccaTaaccca 50 mHAMP (upper) ccTgTcTccTgcTTcTccTccT 51 mHAMP (lower) aaTgTcTgcccTgcTTTcTTcc 52 mHAMP (probe) TgagcagcaccaccTaTcTccaTcaaca 53 TMPRSS6-hcmr-8B 4816151735284816362 54 TMPRSS6-h-2B 3645354745452717261 55 TMPRSS6-hcm-12B 4736264737282646183 56 TMPRSS6-h-7B 3548462715271636271 57 TMPRSS6-hcm-9B 1727354715351718451 58 TMPRSS6-hc-1B 2647364545462646361 59 TMPRSS6-hc-10B 2517363835484518152 60 TMPRSS6-hc-4B 4736454827461736462 61 TMPRSS6-h-3B 3748461727361726351 62 TMPRSS6-h-5B 4517353545171818261 63 TMPRSS6-hc-11B 3847354825464646263 64 TMPRSS6-hc-13B 3615363715372818182 65 TMPRSS6-hcmr-14B 3848453827272535454 66 TMPRSS6-h-6B 2828463647361827163 67 TMPJH01A 6273646282647284546 68 TMPJH01B 1727354715351718451 69 TMPJH02A 2237242282243284142 70 TMPJH02B 1723314715355754815 71 TMPJH03A 2273282646283248182 72 TMPJH03B 5363718351715354815 73 TMPJH04A 2273282646683248182 74 TMPJH05A 2273242242643244542 75 TMPJH05B 5727354315355754455 76 TMPJH06A 2277646242643244542 77 TMPJH07A 2277686242643244542 78 TMPJH08A 2273242246687244542 79 TMPJH09A 2273242682687244542 80 TMPJH10A 2273242242643284586 81 TMPJH11A 2273242242643288586 82 TMPJH12A 2273242246687284586 83 TMPJH13A 6277646246687284586 84 TMPJH13B 5767354315755758855 85 TMPJH14A 2273282282283284182 86 TMPJH14B 5327318315315318415 87 TMPJH15A 2273282282643284182 88 TMPJH16A 2237242242247244582 89 TMPJH16B 1723314355311358411 90 TMPJH17A 2273282242643284586 91 TMPJH18A 6277682242643288142 92 TMPJH19A 6277682242643288586 93 TMPJH20A 2233282242643284142 94 TMPJH21A 2277282242643284586 95 TMPJH22A 2273282642643284586 96 TMPJH23A 2273282282643284586 97 TMPJH24A 2273282246643284586 98 TMPJH25A 2273282242683284586 99 TMPJH26A 2273282242647284586 100 TMPJH27B 5727754715355754855 101 TMPJH28B 1723314311351714411 102 TMPJH29B 5767354315355754455 103 TMPJH30B 5727754315355754455 104 TMPJH31B 5727358315355754455 105 TMPJH32B 5727354315755754455 106 TMPJH33B 5727354315355758455 107 GN-TTR-hc-A ucuugguuacaugaaauccca 108 GN-TTR-hc-B ugggauuucauguaaccaaga 109 GN2-Luc-siRNA 1-A 5(ps)3(ps)826452517383816(ps)3(ps)8 110 GN2-Luc-siRNA 1-B GN2-38163838462527153(ps)8(ps)2 111 STS012-A 6(ps)2(ps)736462826472845(ps)4(ps)6 112 STS012-B GN-17273547153517184(ps)5(ps)1 113 STS012-1-A 6(ps)2(ps)736462826472845(ps)4(ps)6 114 STS012-1-B GN-57677547153517588(ps)5(ps)5 115 STS012-2-A 6(ps)2(ps)736462826472845(ps)4(ps)6 116 STS012-2-B GN-57673543157557588(ps)5(ps)5 117 STS012-3-A 6(ps)2(ps)736462826472845(ps)4(ps)6 118 STS012-3-B GN-57A7C547153517T8G(ps)5(ps)T 119 STS012-4-A 6(ps)2(ps)776462466872845(ps)8(ps)6 120 STS012-4-B GN-57673543157557588(ps)5(ps)5 121 STS012-5-A 6(ps)2(ps)736462426472845(ps)4(ps)6 122 STS012-5-B GN-17273543153517184(ps)5(ps)1 123 STS012-6-A 6(ps)2(ps)73646242647284546(ps)7(ps)7 124 STS012-6-B GN-17273543153517184(ps)5(ps)1 125 STS012-7-A 6(ps)2(ps)776866866472885(ps)8(ps)6 126 STS012-7-B GN-57677547153517588(ps)5(ps)5 127 STS012-8-A 6(ps)2(ps)776866866472885(ps)8(ps)6 128 STS012-8-B GN-57673543157557588(ps)5(ps)5 129 STS012-9-A 2(ps)2(ps)772422422472445(ps)4(ps)2 130 STS012-9-B GN-57277547157557544(ps)5(ps)5 131 control siRNA A 5(ps)1(ps)645262735151828(ps)2(ps)7 132 control siRNA B GN-45353626284515271(ps)6(ps)2 133 hcTMP-SR1-A mCfUmGfAmGfGmAfCmGfCnnCfCmUfGmGfGmAfGmU 134 hcTMP-SR1-B fAmCfUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfG 135 hcTMP-SR2-A mGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmGfAmG 136 hcTMP-SR2-B fCmUfCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfC 137 hcTMP-SR3-A mUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmGfGmA 138 hcTMP-SR3-B fUmCfCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfA 139 hcTMP-SR4-A mGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmGfGmG 140 hcTMP-SR4-B fCmCfCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfC 141 hcTMP-SR5-A mGfGmUfGmCfUmGfAmGfGmAfCmGfCmCfCmUfGmG 142 hcTMP-SR5-B fCmCfAmGfGmGfCmGfUmCfCmUfCmAfGmCfAmCfC 143 hcTMP-SR6-A mGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmCfUmG 144 hcTMP-SR6-B fCmAfGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfC 145 hcTMP-SR8-A mCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmCfCmC 146 hcTMP-SR8-B fGmGfGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfG 147 hcTMP-SR9-A mAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmGfCmC 148 hcTMP-SR9-B fGmGfCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfU 149 hcTMP-SR10-A mUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmCfGmC 150 hcTMP-SR10-B fGmCfGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfA 151 hcTMP-SR11-A mGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmAfCmG 152 hcTMP-SR11-B fCmGfUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfC 153 hcTMP-SR12-A mAfGmUfAmCfGmGfGmGfUmGfCmUfGmAfGmGfAmC 154 hcTMP-SR12-B fGmUfCmCfUmCfAmGfCmAfCmCfCmCfGmUfAmCfU 155 hcTMP-SR13-A mAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmGfGmA 156 hcTMP-SR13-B fUmCfCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfU 157 hcTMP-SR15-A mGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmGfAmG 158 hcTMP-SR15-B fCmUfCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfC 159 hcTMP-SR16-A mGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmUfGmA 160 hcTMP-SR16-B fUmCfAmGfCmAfCmCfCmCfGmUfAmCfUmUfCmCfC 161 hcTMP-SRI7-A mGfCmGfGmAfAmGfUmAfCmGfGmGfGmUfGmCfUmG 162 hcTMP-SR17-B fCmAfGmCfAmCfCmCfCmGfUmAfCmUfUmCfCmCfC 163 hcTMP-SR18-A mUfGmGfGmGfAmAfGmUfAmCfGmGfGmGfUmGfCmU 164 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279 STS12009V11L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmAivG 280 STS12009V11L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfUivG 281 STS12009V29L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivA 282 STS12009V29L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivA 283 STS12009V30L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mAivG 284 STS12009V30L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivG 285 STS12009V34L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 286 STS12009V34L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU 287 STS12009V36L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG(ps2)mA 288 STS12009V36L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU 289 STS12009V37L4-A mA(ps2)fAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 290 STS12009V37L4-B GN2-fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU 291 STS12009V40L4-A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA 292 STS12009V40L4-B GNo-fU(ps2)mCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU(ps2)fU 293 STS12209V4L4-A vinylphosphonate-mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA 294 STS12209V4L4-B GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA 295 STS12209V5L4-A vinylphosphonate-mU fA mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA 296 STS12209V5L4-B GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA 297 STS12209L4-A mU (ps) fA (ps) mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA 298 STS12209L4-6 GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA 299 STS12209V1L4-A mU fA mC fC mA fG mA fA mG fA mA fG mC fA mG fG mU (ps) fG (ps) mA 300 STS12209V1L4-B GN2 fU mC fA mC fC mU fG mC fU mU fC mU fU mC fU mG fG (ps) mU (ps) fA 301 STS18001-A mU(ps)fC(ps)mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA(ps)fC(ps)mG 302 STS18001-B GN2 fCmGfUmAfCmGfCmGfGmAfAmUfAmCfUrnUfC (ps) mG (ps) fA 303 PTEN-2-A caccgccaaaTTTaacTgcaga 304 PTEN-2-B aagggTTTgaTaagTTcTagcTgT 305 PTEN-2-C FAM-TgcacagTaTccTTTTgaagaccaTaaccca-TAMRA 306 hTMSS6: 379U17 ccgccaaagcccagaag 307 hTMSS6: 475L21 ggTcccTccccaaaggaaTag 308 hTMSS6: 416U28FL FAM-cagcacccgccTgggaacTTacTacaac-BHQ1 309 STS12009V54L50-A mA (ps) fA (ps) mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG (ps2) mA 310 STS12009V54L50-B GNo - fU (ps2) mCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 311 STS12009V55L50-A mA (ps) fA (ps) mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG (ps2) mA 312 STS12009V55L50-B GNo - fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 313 STS12009V56L50-A mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA  314 STS12009V56L50-B GNo - fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 315 STS12009V57L50-A mAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG (ps2) mA 316 STS12009V57L50-B GNo - fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU (ps2) fU 317 TMPRSS6-hc-1A augucuuucacacuggcuu 318 TMPRSS6-hc-1B aagccagugugaaagacau 319 TMPRSS6-h-2A auugaguacacgcagacug 320 TMPRSS6-h-2B cagucugcguguacucaau 321 TMPRSS6-h-3A aaguugauggugaucccgg 322 TMPRSS6-h-3B ccgggaucaccaucaacuu 323 TMPRSS6-hc-4A uucuggaucguccacuggc 324 TMPRSS6-hc-4B gccaguggacgauccagaa 325 TMPRSS6-h-5A auucacagaacagaggaac 326 TMPRSS6-h-5B guuccucuguucugugaau 327 TMPRSS6-h-6A guagucauggcuguccucu 328 TMPRSS6-h-6B agaggacagccaugacuac 329 TMPRSS6-h-7A aguuguaguaaguucccag 330 TMPRSS6-h-7B cugggaacuuacuacaacu 331 TMPRSS6-hcmr-8A uuguacccuaggaaauacc 332 TMPRSS6-hcmr-8B gguauuuccuaggguacaa 333 TMPRSS6-hcm-9A aaccagaagaagcagguga 334 TMPRSS6-hcm-9B ucaccugcuucuucugguu 335 TMPRSS6-hc-10A uaacaacccagcguggaau 336 TMPRSS6-hc-10B auuccacgcuggguuguua 337 TMPRSS6-hc-11A guuucucucauccaggccg 338 TMPRSS6-hc-11B cggccuggaugagagaaac 339 TMPRSS6-hcm-12A gcaucuucugggcuuuggc 340 TMPRSS6-hcm-12B gccaaagcccagaagaugc 341 TMPRSS6-hc-13A ucacacuggaaggugaaug 342 TMPRSS6-hc-13B cauucaccuuccaguguga 343 TMPRSS6-hcmr-14A cacagaugugucgaccccg 344 TMPRSS6-hcmr-14B cggggucgacacaucugug 345 TMPRSS6-hcmr-15A uguacccuaggaaauacca 346 TMPRSS6-hcmr-15B ugguauuuccuaggguaca 347 TMPRSS6-Luc-siRNA-1A ucgaaguauuccgcguacg 348 TMPRSS6-Luc-siRNA-1B cguacgcggaauacuucga 349 TMPRSS6-PTEN-A uaaguucuagcuguggugg 350 TMPRSS6-PTEN-B ccaccacagcuagaacuua 351 GN-TTR-hc-A ucuugguuacaugaaauccc a 352 GN-TTR-hc-B ugggauuucauguaaccaag a 353 STS012-6-A aaccagaagaagcaggugacc 354 control siRNA A uuaguaaaccuuuugagac 355 control siRNA B gucucaaaagguuuacuaa 356 hcTMP-SR1-A cugaggacgcccugggagu 357 hcTMP-SR1-B acucccagggcguccucag 358 hcTMP-SR2-A gcugaggacgcccugggag 359 hcTMP-SR2-B cucccagggcguccucagc 360 hcTMP-SR3-A ugcugaggacgcccuggga 361 hcTMP-SR3-B ucccagggcguccucagca 362 hcTMP-SR4-A gugcugaggacgcccuggg 363 hcTMP-SR4-B cccagggcguccucagcac 364 hcTMP-SR5-A ggugcugaggacgcccugg 365 hcTMP-SR5-B ccagggcguccucagcacc 366 hcTMP-SR6-A gggugcugaggacgcccug 367 hcTMP-SR6-B cagggcguccucagcaccc 368 hcTMP-SR8-A cggggugcugaggacgccc 369 hcTMP-SR8-B gggcguccucagcaccccg 370 hcTMP-SR9-A acggggugcugaggacgcc 371 hcTMP-SR9-B ggcguccucagcaccccgu 372 hcTMP-SR10-A uacggggugcugaggacgc 373 hcTMP-SR10-B gcguccucagcaccccgua 374 hcTMP-SR11-A guacggggugcugaggacg 375 hcTMP-SR11-B cguccucagcaccccguac 376 hcTMP-SR12-A aguacggggugcugaggac 377 hcTMP-SR12-B guccucagcaccccguacu 378 hcTMP-SR13-A aaguacggggugcugagga 379 hcTMP-SR13-B uccucagcaccccguacuu 380 hcTMP-SR15-A ggaaguacggggugcugag 381 hcTMP-SR15-B cucagcaccccguacuucc 382 hcTMP-SR16-A gggaaguacggggugcuga 383 hcTMP-SR16-B ucagcaccccguacuuccc 384 hcTMP-SR17-A ggggaaguacggggugcug 385 hcTMP-SR17-B cagcaccccguacuucccc 386 hcTMP-SR18-A uggggaaguacggggugcu 387 hcTMP-SR18-B agcaccccguacuucccca 388 hcTMP-SR19-A cuggggaaguacggggugc 389 hcTMP-SR19-B gcaccccguacuuccccag 390 hcTMP-SR21-A agcuggggaaguacggggu 391 hcTMP-SR21-B accccguacuuccccagcu 392 hcTMP-SR22-A uagcuggggaaguacgggg 393 hcTMP-SR22-B ccccguacuuccccagcua 394 hcTMP-SR23-A guagcuggggaaguacggg 395 hcTMP-SR23-B cccguacuuccccagcuac 396 hcTMP-SR24-A aguagcuggggaaguacgg 397 hcTMP-SR24-B ccguacuuccccagcuacu 398 hcTMP-SR26-A guaguagcuggggaaguac 399 hcTMP-SR26-B guacuuccccagcuacuac 400 hcTMP-SR27-A aguaguagcuggggaagua 401 hcTMP-SR27-B uacuuccccagcuacuacu 402 hcTMP-SR28-A gaguaguagcuggggaagu 403 hcTMP-SR28-B acuuccccagcuacuacuc 404 hcTMP-SR29-A cgaguaguagcuggggaag 405 hcTMP-SR29-B cuuccccagcuacuacucg 406 hcTMP-SR30-A gcgaguaguagcuggggaa 407 hcTMP-SR30-B uuccccagcuacuacucgc 408 hcTMP-SR31-A ggcgaguaguagcugggga 409 hcTMP-SR31-B uccccagcuacuacucgcc 410 hcTMP-SR32-A gggcgaguaguagcugggg 411 hcTMP-SR32-B ccccagcuacuacucgccc 412 hcTMP-SR33-A ggggcgaguaguagcuggg 413 hcTMP-SR33-B cccagcuacuacucgcccc 414 hcTMP-SR34-A uggggcgaguaguagcugg 415 hcTMP-SR34-B ccagcuacuacucgcccca 416 hcTMP-SR35-A uuggggcgaguaguagcug 417 hcTMP-SR35-B cagcuacuacucgccccaa 418 TMPRSS6-hc-16A uauuccaaagggcagcuga 419 TMPRSS6-hc-16B ucagcugcccuuuggaaua 420 TMPRSS6-hc-17A aucuucugggcuuuggcgg 421 TMPRSS6-hc-17B ccgccaaagcccagaagau 422 TMPRSS6-hc-18A uuuucucuuggaguccuca 423 TMPRSS6-hc-18B ugaggacuccaagagaaaa 424 TMPRSS6-hc-19A gaauagacggagcuggagu 425 TMPRSS6-hc-19B acuccagcuccgucuauuc 426 TMPRSS6-hc-21A uaguagcuggggaaguacg 427 TMPRSS6-hc-21B cguacuuccccagcuacua 428 TMPRSS6-hc-22A agauccugggagaaguggc 429 TMPRSS6-hc-22B gccacuucucccaggaucu 430 TMPRSS6-hc-23A cuguucuggaucguccacu 431 TMPRSS6-hc-23B aguggacgauccagaacag 432 TMPRSS6-hcmr-24A cucaccuugaaggacaccu 433 TMPRSS6-hcmr-24B agguguccuucaaggugag 434 TMPRSS6-hcm-25A aguuucucucauccaggcc 435 TMPRSS6-hcm-25B ggccuggaugagagaaacu 436 TMPRSS6-hcr-26A guacccuaggaaauaccag 437 TMPRSS6-hcr-26B cugguauuuccuaggguac 438 TMPRSS6-hc-27A cuguugacuguggacagca 439 TMPRSS6-hc-27B ugcuguccacagucaacag 440 STS12209V4L4-A uaccagaagaagcagguga 441 STS12209V4L4-B ucaccugcuucuucuggua 442 STS18001-A ucgaaguauuccgcguacg 443 STS18001-B cguacgcggaauacuucga 444 PTEN-2-A caccgccaaaTTTaacTgcaga 445 PTEN-2-B aagggTTTgaTaagTTcTagcTgT 446 PTEN-2-C TgcacagTaTccTTTTgaagaccaTaaccca 447 hTMSS6: 379U17 ccgccaaagcccagaag 448 hTMSS6: 475L21 ggTcccTccccaaaggaaTag 449 hTMSS6: 416U28FL cagcacccgccTgggaacTTacTacaac Key 1 = 2′F-dU 2 = 2′dA 3 = 2′F-dC 4 = 2′F-dG 5 = 2′OMe-rU 6 = 2′OMe-rA 7 = 2′OMe-rC 8 = 2′OMe-rG T = dT mA, mU, mC, mG - 2′-OMe RNA fA, fU, fC, fG - 2′-F DNA (ps) - phosphorothioate GN = GalNAc linker GN according to fig. 8A GN2 = GalNAc structure according to fig. 8B GN3 = GalNAc linker structure according to fig. 80 GNo = GN2 with phosphodiesters instead of (ps) [A], [T], [C], [G] - DNA {A}, {U}, {C}, {G} - LNA ivA, ivC, ivU, ivG - inverted RNA (3′-3′) (ps2) - phosphorodithioate vinylphosphonate vinyl-(E)-phosphonate FAM - 6-Carboxyfluorescein TAMRA - 5-Carboxytetramethylrhodamine BHQ - Black Hole Quencher 1

[0795] Where specific linkers and or modified linkages are taught within an RNA sequence, such as PS, PS2, GN, GN2, GN3 etc, these are optional parts of the sequence, but are a preferred embodiment of that sequence.

[0796] The following abbreviations may be used:

TABLE-US-00037 GN [00050] GN2 [00051] GN3 [00052] GNo Same as GN2 but with phosphodiesters instead of phosphorothioates ST23 (building block for synthesis) [00053] ST41/C4XLT (building block for synthesis) [00054] ST43/C6XLT (building block for synthesis) [00055] Long trebler/ltrb/STKS (building block for synthesis) [00056] [ST23 (ps)]3 ST41 GN2 (see above) (ps) [ST23 (ps)]3 ST43 GN3 (see above) (ps) ST23(ps) long GN (see above) trebler(ps)